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Objective: To generate and measure the reliability for a reference standard set with representative cases from seven broad syndromic case definitions and several narrower syndromic definitions used for biosurveillance.Design: From 527,228 eligible patients between 1990 and 2003, we generated a set of patients potentially positive for seven syndromes by classifying all eligible patients according to their ICD-9 primary discharge diagnoses. We selected a representative subset of the cases for chart review by physicians, who read emergency department reports and assigned values to 14 variables related to the seven syndromes.Measurements: (1) Positive predictive value of the ICD-9 diagnoses; (2) prevalence of the syndromic definitions and related variables; (3) agreement between physician raters demonstrated by kappa, kappa corrected for bias and prevalence, and Finn's r; and (4) reliability of the reference standard classifications demonstrated by generalizability coefficients.Results: Positive predictive value for ICD-9 classification ranged from 0.33 for botulinic to 0.86 for gastrointestinal. We generated between 80 and 566 positive cases for six of the seven syndromic definitions. Rash syndrome exhibited low prevalence (34 cases). Agreement between physician raters was high, with kappa > 0.70 for most variables. Ratings showed no bias. Finn's r was >0.70 for all variables. Generalizability coefficients were >0.70 for all variables but three.Conclusion: Of the 27 syndromes generated by the 14 variables, 21 showed high enough prevalence, agreement, and reliability to be used as reference standard definitions against which an automated syndromic classifier could be compared. Syndromic definitions that showed poor agreement or low prevalence include febrile botulinic syndrome, febrile and nonfebrile rash syndrome, respiratory syndrome explained by a nonrespiratory or noninfectious diagnosis, and febrile and nonfebrile gastrointestinal syndrome explained by a nongastrointestinal or noninfectious diagnosis. [ABSTRACT FROM AUTHOR]

A major goal of Natural Language Processing in the public health informatics domain is the automatic extraction and encoding of data stored in free text patient records. This extracted data can then be utilized by computerized systems to perform syndromic surveillance. In particular, the chief complaint--a short string that describes a patient's symptoms--has come to be a vital resource for syndromic surveillance in the North American context due to its near ubiquity. This paper reviews fifteen systems in North America--at the city, county, state and federal level--that use chief complaints for syndromic surveillance., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Standardized surveillance syndromes do not exist but would facilitate sharing data among surveillance systems and comparing the accuracy of existing systems. The objective of this study was to create reference syndrome definitions from a consensus of investigators who currently have or are building syndromic surveillance systems., Design: Clinical condition-syndrome pairs were catalogued for 10 surveillance systems across the United States and the representatives of these systems were brought together for a workshop to discuss consensus syndrome definitions., Results: Consensus syndrome definitions were generated for the four syndromes monitored by the majority of the 10 participating surveillance systems: Respiratory, gastrointestinal, constitutional, and influenza-like illness (ILI). An important element in coming to consensus quickly was the development of a sensitive and specific definition for respiratory and gastrointestinal syndromes. After the workshop, the definitions were refined and supplemented with keywords and regular expressions, the keywords were mapped to standard vocabularies, and a web ontology language (OWL) ontology was created., Limitations: The consensus definitions have not yet been validated through implementation., Conclusion: The consensus definitions provide an explicit description of the current state-of-the-art syndromes used in automated surveillance, which can subsequently be systematically evaluated against real data to improve the definitions. The method for creating consensus definitions could be applied to other domains that have diverse existing definitions.

In this paper we describe an algorithm called ConText for determining whether clinical conditions mentioned in clinical reports are negated, hypothetical, historical, or experienced by someone other than the patient. The algorithm infers the status of a condition with regard to these properties from simple lexical clues occurring in the context of the condition. The discussion and evaluation of the algorithm presented in this paper address the questions of whether a simple surface-based approach which has been shown to work well for negation can be successfully transferred to other contextual properties of clinical conditions, and to what extent this approach is portable among different clinical report types. In our study we find that ConText obtains reasonable to good performance for negated, historical, and hypothetical conditions across all report types that contain such conditions. Conditions experienced by someone other than the patient are very rarely found in our report set. A comprehensive solution to the problem of determining whether a clinical condition is historical or recent requires knowledge above and beyond the surface clues picked up by ConText.

Objective: To determine whether preprocessing chief complaints before automatically classifying them into syndromic categories improves classification performance., Methods: We preprocessed chief complaints using two preprocessors (CCP and EMT-P) and evaluated whether classification performance increased for a probabilistic classifier (CoCo) or for a keyword-based classifier (modification of the NYC Department of Health and Mental Hygiene chief complaint coder (KC))., Results: CCP exhibited high accuracy (85%) in preprocessing chief complaints but only slightly improved CoCo's classification performance for a few syndromes. EMT-P, which splits chief complaints into multiple problems, substantially increased CoCo's sensitivity for all syndromes. Preprocessing with CCP or EMT-P only improved KC's sensitivity for the Constitutional syndrome., Conclusion: Evaluation of preprocessing systems should not be limited to accuracy of the preprocessor but should include the effect of preprocessing on syndromic classification. Splitting chief complaints into multiple problems before classification is important for CoCo, but other preprocessing steps only slightly improved classification performance for CoCo and a keyword-based classifier.

The goals of automated biosurveillance systems are to detect disease outbreaks early, while exhibiting few false positives. Evaluation measures currently exist to estimate the expected detection time of biosurveillance systems. Researchers also have developed models that estimate clinician detection of cases of outbreak diseases, which is a process known as clinical case finding. However, little research has been done on estimating how well biosurveillance systems augment traditional outbreak detection that is carried out by clinicians. In this paper, we introduce a general approach for doing so for non-endemic disease outbreaks, which are characteristic of bioterrorist induced diseases, such as respiratory anthrax. We first layout the basic framework, which makes minimal assumptions, and then we specialize it in several ways. We illustrate the method using a Bayesian outbreak detection algorithm called PANDA, a model of clinician outbreak detection, and simulated cases of a windborne anthrax release. This analysis derives a bound on how well we would expect PANDA to augment clinician detection of an anthrax outbreak. The results support that such analyses are useful in assessing the extent to which computer-based outbreak detection systems are expected to augment traditional clinician outbreak detection.

Objective: Determine whether agreement among annotators improves after being trained to use an annotation schema that specifies: what types of clinical conditions to annotate, the linguistic form of the annotations, and which modifiers to include., Methods: Three physicians and 3 lay people individually annotated all clinical conditions in 23 emergency department reports. For annotations made using a Baseline Schema and annotations made after training on a detailed annotation schema, we compared: (1) variability of annotation length and number and (2) annotator agreement, using the F-measure., Results: Physicians showed higher agreement and lower variability after training on the detailed annotation schema than when applying the Baseline Schema. Lay people agreed with physicians almost as well as other physicians did but showed a slower learning curve., Conclusion: Training annotators on the annotation schema we developed increased agreement among annotators and should be useful in generating reference standard sets for natural language processing studies. The methodology we used to evaluate the schema could be applied to other types of annotation or classification tasks in biomedical informatics.

Evaluating automated indexing applications requires comparing automatically indexed terms against manual reference standard annotations. However, there are no standard guidelines for determining which words from a textual document to include in manual annotations, and the vague task can result in substantial variation among manual indexers. We applied grounded theory to emergency department reports to create an annotation schema representing syntactic and semantic variables that could be annotated when indexing clinical conditions. We describe the annotation schema, which includes variables representing medical concepts (e.g., symptom, demographics), linguistic form (e.g., noun, adjective), and modifier types (e.g., anatomic location, severity). We measured the schema's quality and found: (1) the schema was comprehensive enough to be applied to 20 unseen reports without changes to the schema; (2) agreement between author annotators applying the schema was high, with an F measure of 93%; and (3) the authors made complementary errors when applying the schema, demonstrating that the schema incorporates both linguistic and medical expertise.

Objective: Determine how four contextual features (Validity, Certainty, Directionality, and Temporality) contribute to classification of respiratory syndrome-related clinical conditions as acute, chronic, or absent from manual annotations in Emergency Department Reports. Based on the results, we will direct our research towards automatic identification of the contextual features found to be discriminating., Methods: A physician annotated all instances of 56 clinical conditions in 120 ED reports and encoded four contextual features for every annotation. We classified clinical conditions using the contextual features and measured agreement to reference standard classifications made by the physician using a weighted kappa (Kw)., Results: Kw was 0.518 when not using any of the features and 0.953 when using all of the features., Conclusion: Validity, Directionality, and Temporality all improved accuracy. Negation(Directionality) was the most important feature for improving accuracy. Using Certainty made the classification worse.

Study Objective: Electronic surveillance systems often monitor triage chief complaints in hopes of detecting an outbreak earlier than can be accomplished with traditional reporting methods. We measured the accuracy of a Bayesian chief complaint classifier called CoCo that assigns patients 1 of 7 syndromic categories (respiratory, botulinic, gastrointestinal, neurologic, rash, constitutional, or hemorrhagic) based on free-text triage chief complaints., Methods: We compared CoCo's classifications with criterion syndromic classification based on International Classification of Diseases, Ninth Revision (ICD-9) discharge diagnoses. We assigned the criterion classification to a patient based on whether the patient's primary diagnosis was a member of a set of ICD-9 codes associated with CoCo's 7 syndromes. We tested CoCo's performance on a set of 527,228 chief complaints from patients registered at the University of Pittsburgh Medical Center emergency department (ED) between 1990 and 2003. We performed a sensitivity analysis by varying the ICD-9 codes in the criterion standard. We also tested CoCo on chief complaints from EDs in a second location (Utah)., Results: Approximately 16% (85,569/527,228) of the patients were classified according to the criterion standard into 1 of the 7 syndromes. CoCo's classification performance (number of cases by criterion standard, sensitivity [95% confidence interval (CI)], and specificity [95% CI]) was respiratory (34,916, 63.1 [62.6 to 63.6], 94.3 [94.3 to 94.4]); botulinic (1,961, 30.1 [28.2 to 32.2], 99.3 [99.3 to 99.3]); gastrointestinal (20,431, 69.0 [68.4 to 69.6], 95.6 [95.6 to 95.7]); neurologic (7,393, 67.6 [66.6 to 68.7], 92.7 [92.6 to 92.8]); rash (2,232, 46.8 [44.8 to 48.9], 99.3 [99.3 to 99.3]); constitutional (10,603, 45.8 [44.9 to 46.8], 96.6 [96.6 to 96.7]); and hemorrhagic (8,033, 75.2 [74.3 to 76.2], 98.5 [98.4 to 98.5]). The sensitivity analysis showed that the results were not affected by the choice of ICD-9 codes in the criterion standard. Classification accuracy did not differ on chief complaints from the second location., Conclusion: Our results suggest that, for most syndromes, our chief complaint classification system can identify about half of the patients with relevant syndromic presentations, with specificities higher than 90% and positive predictive values ranging from 12% to 44%.

Objective: Develop and evaluate a natural language processing application for classifying chief complaints into syndromic categories for syndromic surveillance., Introduction: Much of the input data for artificial intelligence applications in the medical field are free-text patient medical records, including dictated medical reports and triage chief complaints. To be useful for automated systems, the free-text must be translated into encoded form., Methods: We implemented a biosurveillance detection system from Pennsylvania to monitor the 2002 Winter Olympic Games. Because input data was in free-text format, we used a natural language processing text classifier to automatically classify free-text triage chief complaints into syndromic categories used by the biosurveillance system. The classifier was trained on 4700 chief complaints from Pennsylvania. We evaluated the ability of the classifier to classify free-text chief complaints into syndromic categories with a test set of 800 chief complaints from Utah., Results: The classifier produced the following areas under the ROC curve: Constitutional = 0.95; Gastrointestinal = 0.97; Hemorrhagic = 0.99; Neurological = 0.96; Rash = 1.0; Respiratory = 0.99; Other = 0.96. Using information stored in the system's semantic model, we extracted from the Respiratory classifications lower respiratory complaints and lower respiratory complaints with fever with a precision of 0.97 and 0.96, respectively., Conclusion: Results suggest that a trainable natural language processing text classifier can accurately extract data from free-text chief complaints for biosurveillance.

Automatic detection of cases of febrile illness may have potential for early detection of outbreaks of infectious disease either by identification of anomalous numbers of febrile illness or in concert with other information in diagnosing specific syndromes, such as febrile respiratory syndrome. At most institutions, febrile information is contained only in free-text clinical records. We compared the sensitivity and specificity of three fever detection algorithms for detecting fever from free-text. Keyword CC and CoCo classified patients based on triage chief complaints; Keyword HP classified patients based on dictated emergency department reports. Keyword HP was the most sensitive (sensitivity 0.98, specificity 0.89), and Keyword CC was the most specific (sensitivity 0.61, specificity 1.0). Because chief complaints are available sooner than emergency department reports, we suggest a combined application that classifies patients based on their chief complaint followed by classification based on their emergency department report, once the report becomes available.

Clinical conditions described in patients' dictated reports are necessary for automated detection of patients with respiratory illnesses such as inhalational anthrax and pneumonia. We applied MetaMap to emergency department reports to extract a set of 71 clinical conditions relevant to detection of a lower respiratory outbreak. We indexed UMLS terms in emergency department reports with MetaMap, filtered the indexed output with a specialized lexicon of UMLS terms for the domain, and mapped the clinical conditions of interest to concepts in the lexicon. We compared MetaMap's ability to accurately identify the conditions against a physician's manual annotations and evaluated incorrectly indexed features to determine what additional processing is necessary. MetaMap identified the clinical conditions with a recall of 0.72 and a precision of 0.56. Necessary processing beyond MetaMap's indexing includes finding validation, temporal discrimination, anatomic location discrimination, finding-disease discrimination, and contextual inference. Successful identification of clinical conditions in an emergency department report with MetaMap requires processing techniques specific to the clinical question of interest.

A large number of biological agents can cause natural or bioterroristic disease outbreaks and each can present in a bewildering number of ways (e.g., a few cases versus many cases, confined to a building versus widely disseminated). This 'problem space' is a challenge for designers of early warning systems for disease outbreaks and the sheer size of this space is a barrier to progress. This paper addresses this problem by deriving nine categories of threats that represent a parsimonious characterization of the problem space. A literature search also identified one or more example outbreaks for each of the nine categories. These outbreaks have occurred in recent times and could be used by researchers in need of actual outbreak data for investigations of the role of different types of surveillance data and algorithms in outbreak detection. The methodological contribution of this research is a Criterion Set of threats for analysis and evaluation of detection systems. This set characterizes the problem space in a tractable manner with less loss of generality than analyses based on one or two selected diseases, which is representative of current analyses.

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.

Hartmannella vermiformis, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of Legionella pneumophila and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of H. vermiformis. H. vermiformis cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with L. pneumophila. Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60 degrees C for 30 min and alternatively to 50 degrees C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55 degrees C, and there were no survivors at 60 degrees C. Pretreatment at 50 degrees C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.

From January 1990 through February 1991, tuberculosis (TB) developed in 10 renal transplant (RT) patients at one hospital; 5 patients died. Possible nosocomial transmission was investigated. Mycobacterium tuberculosis isolates were compared by restriction fragment length polymorphism (RFLP) by a polymerase chain reaction method. The source case occurred in an RT patient (source) who had posttransplant exposure to TB at another hospital. The source patient was rehospitalized on the RT unit; diagnosis of TB and thus isolation precautions were delayed. Epidemiologic and RFLP analysis showed transmission from the source to 5 RT patients and 1 human immunodeficiency virus-infected patient. M. tuberculosis isolates from 4 RT patients had other RFLP patterns. The median incubation period for TB in RT patients was 7.5 weeks (range, 5-11). Bronchoscopy and intubation of the source patient and inadequate ventilation on the RT unit possibly increased transmission. Early detection of TB and effective isolation are essential to prevent nosocomial transmission.

A Flavobacterium meningosepticum outbreak, involving 12 infected and 47 colonized intensive care patients during the months of February through July 1990, was investigated. F. meningosepticum was isolated from tap water and ice, but these environmental strains eventually proved to be distinct from those colonizing patients. A review of newly colonized patients' charts revealed that a common factor among the patients was daily changes of ventilator tubing pasteurized in the hospital's central sterile department. More than 90% of patients in the outbreak had been on ventilators that used the pasteurized tubing. An investigation of the pasteurization process found that two pasteurizer tanks had been operating at suboptimal temperatures (< 62 degrees C). Cultures of water from the tanks and droplets of water found in the pasteurized tubing grew species of Acinetobacter, Moraxella, and Pseudomonas but did not grow F. meningosepticum. After deficiencies in the pasteurization process were corrected, the outbreak terminated. Despite the failure to culture F. meningosepticum, an analysis of gram-negative bacillary isolates showed that the deficiency in the pasteurization process was a major contributor to colonization of ventilated patients by bacteria ubiquitous in tap water.

Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of eukaryotic substrates, including phosphatidylinositol and tubulin. Since the phosphorylation of either phosphatidylinositol or tubulin might compromise phagocyte activation and bactericidal functions, this enzyme may well be a virulence factor. Administration of the L. pneumophila exoprotease induces lesions resembling those of Legionella pneumonia and kills guinea pigs, suggesting that this protein plays a role in the pathogenesis of legionellosis. However, recent work with a genetically engineered strain has convincingly shown that the protease is not necessary for intracellular survival or virulence. As might be expected with a complex process like intracellular parasitism, it appears that the capability of Legionella strains to invade and multiply in host phagocytes is multifactorial and that no single moiety which is responsible for the virulence phenotype will be found.

A model was developed to study the multiplication of various Legionella spp. in tap water containing Hartmannella vermiformis. Tap water cultures prepared with the following components were suitable for the multiplication studies: Legionella spp., 10(3) CFU/ml; H. vermiformis, 10(4.4) cysts per ml; and killed Pseudomonas paucimobilis, 10(9) cells per ml. Cocultures were incubated at 37 degrees C for at least 1 week. The following legionellae multiplied in tap water cocultures in each replicate experiment: L. bozemanii (WIGA strain), L. dumoffii (NY-23 and TX-KL strains), L. micdadei (two environmental strains), and L. pneumophila (six environmental strains and one clinical isolate). Growth yield values for these strains were 0.6 to 3.5 log CFU/ml. Legionellae which did not multiply in replicate cocultures included L. anisa (one strain), L. bozemanii (MI-15 strain), L. micdadei (a clinical isolate), L. longbeachae, (one strain), and L. pneumophila (Philadelphia 1 strain). L. gormanii and an environmental isolate of L. pneumophila multiplied in only one of three experiments. None of the legionellae multiplied in tap water containing only killed P. paucimobilis. The mean growth yield (+/- standard deviation) of H. vermiformis in the cocultures was 1.2 +/- 0.1 log units/ml. H. vermiformis supports multiplication of only particular strains of legionellae, some of which are from diverse origins.

Brucellae are facultative intracellular bacterial pathogens that reside primarily in cells of the reticuloendothelial system. The high-speed supernatant obtained after centrifuging a suspension of Brucella abortus that had been frozen-thawed and sonicated contained abundant phosphomonoesterase activity, determined by using 4-methylumbelliferylphosphate as the substrate; this enzyme was purified 2,900-fold (yield, 570%) by chromatography on DE-52 cellulose and hydroxylapatite columns and high-performance liquid chromatography-gel filtration. The native enzyme had a molecular mass of 120,000 daltons (+/- 10,000 daltons), as determined by gel filtration chromatography, and resolved into two bands (60,000 and 66,000 daltons) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The B. abortus phosphomonoesterase had the following properties: pH optimum, 6.0 to 6.5; isoelectric point, 3.0; substrate specificity, 5'-AMP greater than 3'-AMP greater than 3'-GMP greater than 5'-GDP greater than 5'-CDP greater than 5'-CTP greater than 5'-UPT greater than phosphotyrosine greater than phosphoserine greater than phosphothreonine. The Km for 5'-AMP was 0.37 mM. Phosphatidylinositol 4,5-bisphosphate and myo-inositol 1,3,4-trisphosphate were poor substrates for the B. abortus enzyme. The phosphomonoesterase did not inhibit superoxide anion production by human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine. The phosphomonoesterase may be one of the bacterial enzymes in the pathway leading to the production of adenine, which is secreted by B. abortus and blocks the activation of neutrophils.

The records of 59 immunocompromised patients with fever and pulmonary infiltrates who underwent open lung biopsy, were reviewed. A specific diagnosis was made by lung biopsy in 49 (83%) patients, and in 32 instances (54%) the diagnosis was a treatable infection. Only two (3.4%) false-negative biopsies occurred. Transplant recipients were more likely to have a specific, treatable pneumonia (74%) than patients with a reticuloendothelial malignancy (42%, P less than 0.05). This was due to a greater frequency of bacterial pneumonias, primarily due to Legionella, in transplant recipients (P less than 0.01). However, obtaining a specific diagnosis by lung biopsy did not appear to improve outcome. Seventeen of 32 (53%) patients with treatable infections survived, compared to 8 of 16 (50%) with specific, but untreatable, diagnosis and 6 of 11 (55%) with nondiagnostic biopsies. Advanced age and a low platelet count were predictive of death in both transplant recipients and patients with leukemia and lymphoma (P less than 0.05); a high serum creatinine was an additional predictor in renal transplant recipients.

The susceptibilities of 11 strains representing the five recognized species of Legionella were determined by agar dilution testing on buffered charcoal-yeast extract agar. All of the legionellae tested were susceptible to rifampin, erythromycin, rosaramycin, chloramphenicol, and the aminoglycosides and were resistant to clindamycin and vancomycin. Susceptibilities to penicillins and cephalosporins were variable. Legionella micdadei, Legionella bozemanii, and Legionella gormanii were susceptible to these agents, but minimal inhibitory concentrations for each species were different. Legionella dumoffii resembled Legionella pneumophila in being resistant to penicillin, cephalothin, and cephamandole and susceptible to moxalactam and cefoxitin. All species except L. micdadei produced beta-lactamase.

C3H/He mice chronically infected with murine cytomegalovirus were given skin allografts from histoincompatible BALB/c donors. A significant increase in cytomegalovirus titers occurred within 3 days after placement of the graft in the spleens and kidneys of the allograft recipients as compared with control animals. No significant changes in virus titers were detected in the salivary gland, lung, liver, or blood of allograft recipients. These results indicate that the host-versus-graft reaction alone can enhance murine cytomegalovirus in a chronically infected host and may help explain the high incidence of cytomegalovirus infection seen after renal and other allograft transplantation in man.

A mouse model was used for elucidation of the role of a graft-vs-host reaction in promoting cytomegalovirus infection. F1 (DBA/2 X C3H/He) hybrid mice were infected with murine cytomegalovirus. Five weeks later, a graft-vs.-host reaction was produced in the chronically infected animals by the administration of multiple doses of DBA/2 parental splenocytes. Cytomegalovirus was recovered more often from the organs of mice undergoing graft-vs.-host reaction than from those of control animals (P less than 0.001). These results indicate that the graft-vs.-host reaction alone can enhance murine cytomegalovirus infection in a chronically infected host and may help explain the high incidence of cytomegalovirus infection after bone marrow allograft transplantation in man.

Two children with legionellosis complicating a relapse of acute lymphoblastic leukemia are reported. A 5-year-old boy with pneumonia had Legionella pneumophila cultured from a tracheal aspirate following a rapid deterioration in his respiratory status and intubation. This child had severe and irreversible granulocytopenia and died in spite of therapy with erythromycin and rifampin added five days later. Combination antimicrobial therapy is suggested for immunosuppressed children with legionellosis if resolution of neutropenia is not readily anticipated. Culture of Legionella sp from respiratory tract secretions or sputum, as reported for the first time in the pediatric literature, should be attempted in all children in whom this infection is suspected. A 13-year-old boy with pneumonia recovered in spite of therapy with antimicrobial agents not proven to be effective against the legionellae. Clinical improvement coincided with increase in absolute granulocyte count. A retrospective diagnosis was made when seroconversion to Legionella micdadei (less than 1:16 to 1:1,024) was determined during a survey of unselected sera from 255 hospitalized children. This is the first documented case of Pittsburgh pneumonia described in a child.

A patient had disseminated herpes simplex, type 1, virus infection manifested by fulminant hepatitis and disseminated intravascular coagulation. The diagnosis was established by isolation of the virus from throat, urine, and buffy coat and confirmed at autopsy by the visualization of typical inclusions, demonstration of herpesvirus particles by electron microscopy, and specific immunoperoxidase staining. Therapy with vidarabine did not alter the fatal course. On the basis of clinical features and serologic results, the case represented a disseminated primary infection with herpes simplex, rather than reactivation of an endogenous infection, following renal transplantation.

Eight immunosuppressed patients had pneumonia due to Pittsburgh Pneumonia Agent (PPA), a gram-negative, weakly acid-fast bacterium cultivatable only in embryonated eggs and guinea pigs and distinct from Legionella pneumophila. The diagnosis was established by isolation of the agent from lung or visualization of the organism in lung tissue. The clinical presentations, radiographic abnormalities and pathology were not specific. The most consistent feature associated with the disease was the recent institution of daily high-dose corticosteriod therapy in all patients. Five of the eight patients died despite broad-spectrum antibiotic and antituberculous therapy. Anti-microbial activity against PPA was demonstrated for sulfamethoxazole combined with trimethoprim, for rifampin and for erythromycin with an egg-protection assay. Serologic studies with an indirect fluorescent-antibody technic suggested that seroconversion or high titers may be a sensitive test for PPA disease. PPA appears to be a newly recognized cause of life-threatening bacterial pneumonia in immunosupressed patients.

A group of 124 children undergoing open-heart surgery was followed prospectively in order to estimate the risk of cytomegalovirus (CMV) infection due to transfused blood.Ninety-three patients (75%) had complement fixation (CF) titers of < 1:4 against CMV on admission. Of this seronegative subgroup, nine patients (9.7%) subsequently became infected with CMV. All nine showed seroconversion, and six were viruric 12-14 weeks after surgery. Comparative seroepidemiological studies of the hospital population showed that in the age ranges studied (3-16 yr), the infections seen in the study group represented a significant excess over expectation. This infection rate was consistent with a model of transmission by blood transfusion with a risk of 2.7% per unit but not proven.Thirty-one patients had CF antibody to CMV on admission. CMV was isolated from 14% of urines of seropositive children both before and after surgery, but only two patients showed CF antibody rises to CMV. Thus the frequency of CMV infection associated with open-heart surgery and transfusion could not be calculated in the seropositive subgroup.CMV infection was not related to the primary diagnosis or to Down's syndrome.

Pittsburgh pneumonia agent (Legionella micdadei) grew in monkey, chicken, and human cell cultures. Pittsburgh pneumonia agent grew predominantly in the cytoplasm, resulting in a nonfocal, mild cytopathic effect.

The interaction of Legionella micdadei with human polymorphonuclear neutrophils (PMNs) and serum was investigated to determine their roles in host defense against this organism. Serum and PMNs from normal donors having no antibody to L micadei were used. PMNs phagocytized once-agar-passaged (74.6% +/- 6.5%) and twice-agar-passaged (87.3% +/- 1.0%) L micdadei less (P less than 0.05) than L micdadei passaged multiple times on agar (97.5% +/- 1.0%) or Staphylococcus aureus (98.3% +/- 0.5%). Under the same conditions, no phagocytosis of Legionella pneumophila occurred. Use of heat-inactivated serum abolished phagocytosis. PMN killing of once-agar-passaged (1.5% +/- 1.5%) and twice-agar-passaged (0) L micdadei was less (P less than 0.001) than that of L micdadei passaged multiple times on agar (88.6% +/- 4.0%) or S aureus (96.8% +/- 0.5%). L micdadei was not killed by fresh serum, although, in contrast to L pneumophila, it was opsonized by C3. Thus virulent L micdadei is phagocytized, but not killed, by human PMNs, with complement being the major opsonin.

An immunocompromised patient developed pneumonia in which both Legionella micdadei and L. pneumophila, serogroup 6, were isolated from transtracheal aspirates in the absence of any other bacteria. Unusual features included the development of disseminated intravascular coagulation and multiple pulmonary abscesses during treatment with erythromycin, and relapse of the legionellosis after extended therapy with both erythromycin and rifampin, which was heralded by septic shock. Because simultaneous infection with two distinct species of Legionella occurs, concurrent increases in antibody titer to more than one Legionella may, in some instances, reflect multiple infection rather than cross-reacting antibody.

Samples obtained from plumbing systems of hospitals, nonhospital institutions and homes were cultured for Legionella spp. by plating the samples directly on a selective medium. Swab samples were taken from the inner surfaces of faucet assemblies (aerators, spouts, and valve seats), showerheads, and shower pipes. Water and sediment were collected from the bottom of hot-water tanks. Legionella pneumophila serogroups 1, 5, and 6 were recovered from plumbing fixtures of the hospitals and nonhospital institutions and one of five homes. The legionellae (7 to 13,850 colony-forming units per ml) were also present in water and sediment from hot-water tanks maintained at 30 to 54 degrees C, but not in those maintained at 71 and 77 degrees C. Legionella micdadei was isolated from one tank. Thus legionellae are present in hot-water tanks which are maintained at warm temperatures or whose design results in warm temperatures at the bottom of the tanks. We hypothesize that hot-water tanks are a breeding site and a major source of L. pneumophila for the contamination of plumbing systems. The existence of these bacteria in the plumbing systems and tanks was not necessarily associated with disease. The extent of the hazard of this contamination needs to be delineated.

Human cell culture-derived interferon was shown to inhibit human cytomegalovirus in vitro. A prototype strain, Davis, and six clinical isolates of cytomegalovirus were tested. All six isolates showed uniform susceptibility to interferon, exceeding that of the Davis strain by two- to fourfold. The latter virus was found to be 32 to 4 times less susceptible than the sensitive indicator, vesicular stomatitis virus. However, the laboratory finding of susceptibility to an antiviral material may not relate to its clinical effectiveness.

Several antimicrobial agents were evaluated for activity against experimental Legionella micdadei pneumonia in guinea pigs. Erythromycin, rifampin, doxycycline, and sulfamethoxazole-trimethoprim produced significant reductions in mortality. Penicillin, cefazolin, cefoxitin, chloramphenicol, and gentamicin were not efficacious even though, at the doses administered, the peak concentrations of these agents in serum substantially exceeded their MICs for the test strain. It is suggested that the poor performance of the latter group of agents resulted from poor penetration into cells in which L. micdadei was multiplying.

To determine the incidence of cytomegalovirus infection in renal-transplant recipients we followed 32 prospectively for six months after operation. As judged by serologic change and virus isolation the infection rate for the entire group was 66 per cent (21 of 32 patients) - 59 per cent (13 of 22) for seronegative patients and 80 per cent (eight of 10) for seropositive patients. Of 10 seronegative patients who received kidneys from seronegative donors, only three became infected. However, of 12 seronegative patients who received kidneys from seropositive donors, 10 became infected. Thus, there was a significant correlation between development of infection and seropositivity of the donor (P = 0.03), particularly when the recipient was seronegative (P = 0.02). Five possible and four definite recognizable clinical illnesses were associated with cytomegalovirus infection; all except two were in initially seronegative subjects who received kidneys from seropositive donors. Primary infection and disease in nonimmune recipients may be caused by cytomegalovirus transmitted by the kidneys of latently infected seropositive donors.

Twenty acutely ill patients requiring prolonged orotracheal intubation were studied to determine the source and progression of gram-negative bacilli colonizing the trachea. Organisms recovered from daily tracheal, hypopharyngeal, and rectal cultures were typed and speciated to identify identical strains at the three sites. All patients acquired gram-negative bacilli in the trachea by day 3 after intubation. Thirty organisms that were not recovered from the tracheal aspirate immediately following intubation were isolated for at least two days some time thereafter. Nine of the 30 colonizing bacteria were Enterobacteriaceae, and all were found in another culture site, usually the hypopharynx, before isolation from the trachea. In contrast, only four of the 21 non-Enterobacteriaceae that colonized the trachea were recovered previously from either the hypopharynx or rectum, a finding which represents a significant difference (P = 0.0002). Quantitation of isolates from the hypopharynx was of no value in predicting subsequent acquisition in the trachea, and the numbers of bacteria recovered from the first positive tracheal specimen were not predictive of subsequent persistence in the trachea.

Legionella micdadei, a pathogen which enters into host phagocyte phagolysosomal structures, contains at least two protein kinases. We have purified to homogeneity the predominant, nucleotide-independent protein kinase and examined its ability to catalyze the transfer of phosphate from ATP to acceptors in human neutrophils. The L. micdadei protein kinase catalyzed the phosphorylation of proteins of 11.5, 14, 19, 23, 28, 34, and 38 kilodaltons (kDa) present in a Triton X-100 extract of neutrophil membranes and of 11.5, 13.5, 25, and 38 kDa in the neutrophil cytosol. Tubulin was a good substrate for the L. micdadei protein kinase in vitro. The bacterial kinase also catalyzed the phosphorylation of phosphatidylinositol (PI) at about half the rate at which histones were phosphorylated; phosphatidylinositol-4-phosphate (PIP) was not phosphorylated by the kinase. The PI kinase activity of the L. micdadei enzyme was optimum at pH 7.0, and the divalent cation requirement was satisfied best by Mg2+ and Ca2+. The maximum rate of PI phosphorylation was obtained with 0.6 mM PI; in the presence of MgCl2 (10 mM), the Km for PI was 0.9 mM and the Km for ATP was 1.5 mM. The detergents octyl-beta-D-glucoside (10 to 20 mM) and Triton X-100 (0.5%) stimulated kinase activity twofold when PI was the phosphate acceptor; however, only octyl glucoside stimulated histone kinase activity. Various membrane phospholipids inhibited PI kinase activity. The most potent phospholipid inhibitor was the product of the PI kinase reaction, PIP, which at a 0.6 mM concentration inhibited both PI and tubulin phosphorylation by 80%. The inhibition of kinase activity by PIP when histone served as the acceptor was noncompetitive in character. The L. micdadei kinase also phosphorylated PI in intact. (3H)inositol-labeled neutrophils. The PI kinase and histone kinase activities of teh L. micdadei kinase copurified and cofucused (pI, 5.8) when subjected to isoelectric focusing, suggesting that the two enzymatic activities reside in a single protein.

Ribavirin (1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxamide), a new synthetic nucleoside, inhibited murine cytomegalovirus in cell culture. This was shown by the inhibition of viral cytopathic effect and plaque formation, as well as reduction in the yield of virus. Despite this in vitro antiviral effect, ribavirin did not protect mice from mortality produced by a high inoculum of cytomegalovirus. When a lower inoculum was used to initiate a chronic infection, the administration of ribavirin for 9 days had no effect on the titer of cytomegalovirus in the salivary gland, kidney, liver, and spleen. Thus, ribavirin was ineffective in the treatment of both acute and chronic murine cytomegalovirus infections.

We have recently shown that Legionella micdadei is ingested, but not killed, by human neutrophils. Herein we investigate the role of human monocytes in defense against this organism. Serum and monocytes from normal donors having no detectable antibody to L. micdadei were used. Egg-passaged L. micdadei organisms multiplied inside these monocytes with a peak growth of 2 log units within 12 h. No growth occurred when monocytes were omitted or when sonicated monocytes were used. Electron microscopy 18 h after infection revealed these organisms to be intracellular in normal-appearing phagosomes. When the input multiplicity of L. micdadei was greater than 1 CFU per monocyte, no intracellular growth occurred. When egg-passaged Legionella pneumophila organisms were used, intracellular organisms were found in phagosomes studded with ribosomes at the same time period. Peak intracellular growth of L. pneumophilia occurred by 48 h. L. micdadei activated the complement system and was opsonized by C3. However the use of complement-depleted (heat-inactivated) serum as the opsonic source had no effect on the bacterium's ingestion or growth in the monocyte. Thus, L. micdadei multiples in human monocytes. This entry and growth is independent of antibody or complement. The intracellular locations of L. micdadei and L. pneumophila differ, suggesting different mechanisms for the survival of these two organisms in the monocyte.

Protein kinases I (PK I) and II (PK II) were purified 253- and 13.5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a Km of 2.7 mg ml-1 for PK I and 2.9 mg ml-1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6.8 and 7.0 for PK I and PK II respectively. The Km for ATP was 0.29 mM for PK I and 0.33 mM for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.

Guinea pigs develop a lethal pneumonia after intratracheal infection with Legionella micdadei, and the lung displays pathological changes similar to those observed in humans. To investigate the role of the resident alveolar macrophage in the pathogenesis of L. micdadei pneumonia, guinea pig alveolar macrophages obtained by bronchoalveolar lavage were cultured in vitro and infected with L. micdadei. In the absence of opsonins L. micdadei was phagocytized by, and multiplied within, alveolar macrophages with greater than a 100-fold increase in cell-associated colony forming units over 20 h. L. micdadei opsonized with complement or antibody multiplied within alveolar macrophages at the same rate as unopsonized bacteria. Guinea pigs which were treated with antimicrobials after infection with L. micdadei and recovered from the pneumonia were immune to challenge with an otherwise lethal inoculum of L. micdadei. However, the growth curve of both unopsonized and opsonized L. micdadei in the alveolar macrophages from immune animals was essentially identical to that in macrophages from susceptible animals. Thus, the resident alveolar macrophage is not capable of limiting the growth of Legionella. Rather, the alveolar macrophages appear to be the primary site of Legionella multiplication within the lung. Although alveolar macrophages may participate in other aspects of pulmonary immunity to the legionellae, these data indicate that the alveolar macrophage alone does not act as an effector cell in cell-mediated immunity to Legionella.

The high-speed supernatant (100,000 g, 1 h) obtained after centrifuging a suspension of Legionella micdadei that had been freeze-thawed and sonicated contained (i) considerable acid phosphatase activity when assayed using 4-methylumbelliferyl phosphate (MUP) as the substrate, and a factor that blocked superoxide anion production by human neutrophils stimulated with f-Met-Leu-Phe. Chromatography of the extract on a hydroxylapatite column resolved two acids phosphatases (designated ACP1 and ACP2). Subsequent chromatography of ACP2 on a Sephadex G-150 column revealed coincident elution of phosphatase activity and neutrophil blocking activity. When heated at 45 degrees C for various periods of time, the phosphatase activity of the acid phosphatase preparation was lost at the same rate as the ability of the preparation to block superoxide anion production by neutrophils. Furthermore, preincubation of neutrophils and acid phosphatase together in the presence of a heteropolymolybdate complex that inhibits the phosphatase eliminated the effect of the L. micdadei phosphatase on neutrophil superoxide anion production. ACP2 had the following properties: pH optimum, 6.0; Km for MUP, 3.8 mM; isoelectric point, 4.5; substrate specificity, MUP greater than ADP greater than phosphoenolpyruvate greater than phosphothreonine greater than phosphoserine greater than phosphotyrosine; molecular weight (estimated by sucrose density gradient centrifugation and gel filtration chromatography), 71,000-86,000. These results indicate that a cell-associated phosphatase may play a role in the virulence of L. micdadei.

The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself.

Erythromycin-resistant Legionella spp. variants were obtained by a single passage of the naturally occurring bacteria on medium containing various concentrations of erythromycin. By disk diffusion susceptibility testing, at least three different phenotypic patterns of cross-resistance to macrolide, lincosamide, and streptogramin B antibiotics were observed among the 26 erythromycin-resistant strains examined.