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PubMed1994

Bronchoscopic diagnosis of pneumonia.

Lower respiratory tract infections are characterized by significant morbidity and mortality but also by a relative inability to establish a specific etiologic agent on clinical grounds alone. With the recognized shortcomings of expectorated or aspirated secretions toward establishing an etiologic diagnosis, clinicians have increasingly used bronchoscopy to obtain diagnostic samples. A variety of specimen types may be obtained, including bronchial washes or brushes, protected specimen brushings, bronchoalveolar lavage, and transbronchial biopsies. Bronchoscopy has been applied in three primary clinical settings, including the immunocompromised host, especially human immunodeficiency virus-infected and organ transplant patients; ventilator-associated pneumonia; and severe, nonresolving community- or hospital-acquired pneumonia in nonventilated patients. In each clinical setting, and for each specimen type, specific laboratory protocols are required to provide maximal information. These protocols should provide for the use of a variety of rapid microscopic and quantitative culture techniques and the use of a variety of specific stains and selective culture to detect unusual organism groups.

Bronchoscopy↗
PubMed1990

Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.

Alginate, a viscous polysaccharide from mucoid Pseudomonas aeruginosa, may interfere with the host defenses in patients with cystic fibrosis and chronic P. aeruginosa lung infection. The alginate concentration in the sol phase of expectorated sputum was quantitated by a biochemical method and a newly developed enzyme-linked immunosorbent assay. There was a high degree of correlation between the methods, and the concentration of alginate ranged from 4 to 101 micrograms/ml with a median of 35.5 micrograms/ml when measured by enzyme-linked immunosorbent assay. Alginate could not be detected in the bronchial secretions from patients without P. aeruginosa infection. In vitro investigation of alginate did not show any activation of the alternative pathway of complement, as determined by a hemolytic kinetic assay and by testing for neutrophil chemotaxis. At a high concentration, P. aeruginosa alginate caused a slight activation of the classical pathway of complement. Alginate did not cause neutrophil chemotaxis by itself but was able to reduce the neutrophil chemotactic response to N-formylmethionylleucylphenylalanine and for zymosan-activated serum. P. aeruginosa and seaweed alginates were able to prime neutrophils for increased N-formylmethionylleucylphenylalanine-induced neutrophil oxidative burst, as determined by chemiluminescence. Because of its ability to prevent attraction of neutrophils to the site of infection, lack of complement activation, and ability to enhance neutrophil oxidative burst, alginate from P. aeruginosa may contribute to the persistence and pathogenesis of chronic P. aeruginosa infection in cystic fibrosis.

Alginates↗
PubMed1980

Direct immunofluorescent detection of Legionella pneumophila in respiratory specimens.

Respiratory secretions from patients with clinically suspected Legionnaires pneumonia were examined by direc immunofluorescent tests at the Medical Center Hospital of Vermont and at the Center for Disease Control. No fluorescent bacteria were found by either laboratory in eight specimens from eight patients who were seronegative. Twenty specimens were obtained from seven patients who had serologically confirmed Legionnaires disease. Four of the seven cases were identified at the Medical Center Hospital of Vermont, and six of the seven were identified at the Center for Disease Control. Of 20 specimens, 8 were positive at the Center for Disease Control (six or more bacilli per slide), and 7 specimens were suspicious (one to five bacilli per slide); at the Medical Center Hospital of Vermont, 4 of 20 specimens were positive, and 2 were suspicious. The inclusion of a rhodamine-conjugated counterstain at the Center for Disease Control facilitated the examination and may have improved the sensitivity. Smears from transtracheal aspirates, bronchoscopic aspirates, transcutaneous lung aspirates, pleural fluids, and tracheal aspirate-expectorated sputum produced positive results. Several specimens contained fluorescing bacilli when stained for serogroup 2 as well as serogroup 1, perhaps reflecting the presence of cross-reacting antigens in vivo.

Bacteria↗
PubMed1980

Misdiagnosis caused by fungal contaminant in a histological stain solution.

Silver stains on tissue and cytology specimens are important in the evaluation of patients with suspected fungal infections. Care must be taken, however, to prevent misinterpretation of contamination artifacts. Two cases presenting such a problem are reported. The first patient had granulomatous leg lesions that microscopically showed characteristics of erythema induratum but with budding yeastlike organisms demonstrated by Grocott methenamine silver stain. Cultures and subsequent biopsies were negative for fungi. The second patient had a steroid-dependent chronic obstructive lung disease, and during evaluation for possible Pneumocystis carinii pneumonia, the Grocott methenamine silver stain on expectorated sputum showed budding yeastlike organisms. Sputum cultures were negative for fungi. Examination of the two Grocott light-green counterstain solutions demonstrated black, budding yeast cells similar to those seen in the specimens from the patients. Culture of the counterstain grew Exophiala (Phialophora) jeanselmei. Further studies revealed that this cause of misdiagnosis could be prevented by either filtering or adding thymol to the counterstain solution. Care regarding contamination of histological stain solutions is emphasized.

Granuloma↗
PubMed1982

Comparison of six different criteria for judging the acceptability of sputum specimens.

A series of 391 unselected expectorated sputum specimens was examined microscopically, and six different published criteria for judging the acceptability of the specimens were applied. Of the 391 specimens, 234 were found to be acceptable or unacceptable by all six criteria; 157 specimens were discrepant. By the criteria of Murray and Washington and of Barry, 25 and 23% of the specimens, respectively, were rejected; only 19 of 143 specimens which contained potential pathogens as part of their predominant microbial flora were rejected by both sets of criteria. The criteria described by Geckler et al. and Bartlett missed fewer potential pathogens; only 9 or 17% of the specimens, respectively, were found unacceptable. The criteria of Heineman and Radano and of Van Scoy resulted in the greatest percentages of specimens judged unacceptable (28 and 29%, respectively), including 19 and 24% of specimens containing potential pathogens. The reproducibility of sputum screening results was also assessed, comparing the method of Murray and Washington with that of Barry. Six separate slides were prepared from each of 45 different specimens: three samples with purulent or bloody flecks and three samples in which the specimens had been mixed with an applicator swab. Satisfactory reproducibility was observed with both criteria and both sampling methods; no significant differences in reproducibility could be documented in this limited series.

Bacteria↗
PubMed1987

Bordetella bronchiseptica bronchitis.

The clinical course of a patient with bronchitis caused by Bordetella bronchiseptica is described. The organism was recovered on one occasion from a protected catheter brush specimen obtained at bronchoscopy and on two occasions from expectorated sputum specimens. The infection was eradicated with antimicrobial therapy.

Bordetella↗
PubMed1987

Nonvalue of sputum culture in the management of lower respiratory tract infections.

Establishment of the microbiological etiology of bacterial pneumonia by sputum culture is confounded by both lack of recovery of fastidious pathogens and contamination of specimens with oropharyngeal flora. We reviewed the clinical records from 249 patients over a 3-month period for evidence of pneumonia. Gram staining and cultures were performed on 381 specimens isolated from this population of patients. Recovery of respiratory tract pathogens was accomplished with 354 specimens from 226 patients; 27 specimens yielded normal flora in culture but were smear positive. An additional 256 specimens submitted to our microbiology laboratory did not meet smear criteria for purulence nor did they yield respiratory tract pathogens in culture. A total of 637 specimens submitted to the microbiology laboratory were evaluated for sputum purulence by the criteria of Bartlett. Of the total 354 specimens which were positive in culture for a pathogen, 182 (52%) were submitted from 150 patients with no objective evidence of pneumonia. The majority of specimens obtained from patients without pneumonia were nonpurulent. However, 71 of 182 culture-positive specimens obtained from 50 patients without pneumonia were purulent. Approximately half of these patients (31 of 50) had other pulmonary or upper respiratory tract pathology which could account for the sputum purulence. Among the 172 culture-positive specimens from 76 patients with pneumonia, only 100 (58%) were acceptable by smear criteria. An additional 23 patients provided expectorated purulent sputum from which no respiratory tract pathogen could be isolated. Of these 23, 7 had pneumonia. We conclude that sputum culture and Gram staining are neither specific nor sensitive as diagnostic tools. Objective criteria for purulence of Gram-stained specimens must be applied before their inoculation into culture media. Specimens should be sought only from patients with objective evidence of pneumonia.

Bacteria↗
PubMed1988

Sputum gram stain assessment in community-acquired bacteremic pneumonia.

A prospective study was performed over a 4.5-year period to determine the ability of a sputum Gram stain to predict the cause of community-acquired bacterial pneumonia. A blood culture isolate, rather than a sputum culture, served as the reference standard to provide precise identification of the etiologic agent. The study population comprised 59 bacteremic adults who expectorated a valid sputum sample. Data are presented that indicate that a physician, aided by the morphology of the stained sputum, could theoretically select appropriate monotherapy approximately 94% of the time when selective, defined criteria for the microbiology of valid sputum are met. Three of the five patients with pneumonia caused by Haemophilus influenzae, however, had sputum stains that suggested alternative pathogens. This study reaffirms that the Gram-stained sputum is a reliable, but not infallible, guide to direct initial antibiotic therapy in adults with community-acquired bacterial pneumonia.

Adolescent↗
PubMed1989

Activities and sources of beta-lactamase in sputum from patients with bronchiectasis.

beta-Lactamase activity was measured in secretions from patients with bronchiectasis. Of 28 sputum samples, 23 contained measurable amounts of activity; values were significantly higher (P less than 0.01) in purulent samples than in mucoid or mucopurulent samples. beta-Lactamase activity was usually present in saliva collected before and between sputum expectorations, although values for sputum were higher than for either group of saliva samples (P less than 0.025 and P less than 0.005, respectively). This difference suggests that at least part of sputum beta-lactamase activity originates in the bronchial tree. Detailed microbiological study of a further eight specimens (seven were beta-lactamase positive) led to the isolation of Haemophilus influenzae from six, although only two of these isolates were beta-lactamase positive. Several other beta-lactamase-producing organisms were also isolated, including Staphylococcus aureus (n = 3), Escherichia coli (n = 1), Proteus spp. (n = 1), and Bacteroides spp. (n = 3). Size-exclusion high-performance liquid chromatography of the sputum showed several peaks of beta-lactamase activity which usually coeluted in fractions similar to those of their beta-lactamase-positive isolates. Therefore, sources of sputum beta-lactamases are often bacteria not considered truly pathogenic or not isolated during routine bacteriological assessment. These observations should be considered when embarking on antimicrobial therapy in bronchiectatic patients and suggest that increased dosages of penicillins are indicated.

Albumins↗
PubMed1992

Quantitation of pneumococcal C polysaccharide in sputum samples from patients with presumptive pneumococcal pneumonia by enzyme immunoassay.

Although the Gram stain and culture of expectorated sputum are considered standard methods for the diagnosis of presumptive pneumococcal pneumonia, these methods remain relatively insensitive and nonspecific. We developed an enzyme immunoassay (EIA) for the quantitation of pneumococcal C polysaccharide (PnC) in the sputum of patients with presumptive pneumococcal pneumonia. Of 34 patient sputum samples collected within 24 h of the first radiographic report of pneumonia, 12 grew Streptococcus pneumoniae on culture. By using a cutoff point of 0.5 micrograms of PnC per ml of sputum, all 12 specimens were positive (sensitivity, 100%) by EIA. PnC levels ranged from 1.43 to 57.53 micrograms/ml. Blood samples from 18 of the 34 patients were cultured. S. pneumoniae grew in the culture of a blood sample from one patient, whose sputum also had the highest PnC level. Of 22 sputum samples from patients with pneumonia that did not grow S. pneumonia, two were positive by EIA (specificity, 90.1%). Sputa from both patients had low levels of PnC (2.7 and 4.5 micrograms/ml), and both patients had received antibiotics before sputum collection. The positive predictive value of the quantitative EIA was 85.7%. Quantitation of PnC has the potential for improving the accuracy of sputum examination for S. pneumoniae, monitoring disease severity and the effectiveness of antibiotic therapy, and differentiating between those patients with invasive pneumococcal disease and those who are carriers of S. pneumoniae.

Bacteriological Techniques↗
PubMed1995

Comparison of PCR and standard cytological staining for detection of Pneumocystis carinii from respiratory specimens from patients with or at high risk for infection by human immunodeficiency virus.

The detection of Pneumocystis carinii DNA by PCR was compared with routine cytologic staining techniques (CYT). A total of 284 clinical respiratory specimens, including 137 bronchoalveolar lavage (BAL), 63 bronchoalveolar washing, 63 sputum, and 21 induced sputum samples, obtained from patients with or at high risk for human immunodeficiency virus infection were evaluated. Eighty specimens were positive by PCR, and 69 were positive by CYT. PCR was able to detect P. carinii in more bronchoalveolar washing specimens (15 versus 11) and in comparable BAL specimens (53 versus 54) compared with CYT. PCR was particularly more sensitive than CYT in detecting P. carinii in expectorated sputum (12 versus 4 samples). Of the 19 patients whose respiratory specimens were positive for P. carinii by PCR but negative by CYT, 5 had P. carinii pneumonia (PCP) confirmed by subsequent BAL and transbronchial or mediastinal lymph node biopsy and 9 had a clinical course highly suggestive of acute PCP. Eleven (58%) of the 19 patients with discordant PCR and CYT results had received prior anti-PCP prophylaxis. In this clinical setting in particular and in the evaluation of sputum specimens, the ability of PCR to detect a low parasitic load suggests that this technique may become an important additional tool, along with current cytological methods, for the detection of P. carinii.

Adult↗
PubMed1999

False-positive Gen-Probe direct Mycobacterium tuberculosis amplification test results for patients with pulmonary M. kansasii and M. avium infections.

The Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) test has been approved for use in the United States for the rapid diagnosis of pulmonary tuberculosis in patients with acid-fast smear-positive sputum samples since 1996. Four patients infected with human immunodeficiency virus and one chronic pulmonary-disease patient seen in our institutions with abnormal chest radiographs and fluorochrome stain-positive sputa were evaluated for tuberculosis, including performance of the MTD test on expectorated sputum samples. Three of these five patients' sputa were highly smear-positive (i.e. , more than 100 bacilli per high-power field), while two patient's sputa contained 1 to 10 bacilli per field. MTD results on sputum specimens from these patients ranged from 43,498 to 193,858 relative light units (RLU). Gen-Probe has defined values of at least 30,000 RLU as indicative of a positive test, i.e., the presence of Mycobacterium tuberculosis RNA. Four of the patients' sputum cultures yielded growth of M. kansasii within 6 to 12 days, and the fifth produced growth of M. avium only. One patient's culture contained both M. kansasii and M. avium, but none of the initial or follow-up cultures from these five patients revealed M. tuberculosis. However, subsequent cultures from three of the patients again revealed M. kansasii. During the period of this study, in which MTD tests were performed on smear-positive sputum specimens from 82 patients, four of seven patients with culture-proven M. kansasii pulmonary infections yielded one or more false-positive MTD tests. The MTD sensitivity observed in this study was 93.8%, and the specificity was 85.3%. Five cultures of M. kansasii (including three of these patients' isolates and M. kansasii ATCC 12478), and cultures of several other species were examined at densities of 10(5) to 10(7) viable CFU/ml by the MTD test. All five isolates of M. kansasii and three of three isolates of M. simiae yielded false-positive test results, with readings of 75,191 to 335,591 RLU. These findings indicate that low-level false-positive MTD results can occur due to the presence of M. kansasii, M. avium, and possibly other Mycobacterium species other than M. tuberculosis in sputum. Low-level positive MTD results of 30,000 to 500,000 RLU should be interpreted in light of these findings. It remains to be determined if the enhanced MTD test (MTD 2) recently released by Gen-Probe will provide greater specificity than that observed in this report with its first-generation test.

AIDS-Related Opportunistic Infections↗
PubMed2000

Increased sensitivity of the BACTEC 460 mycobacterial radiometric broth culture system does not decrease the number of respiratory specimens required for a definitive diagnosis of pulmonary tuberculosis.

The BACTEC 460 radiometric mycobacterial broth culture system has consistently demonstrated faster and increased recovery of Mycobacterium tuberculosis from respiratory specimens of patients with pulmonary tuberculosis than conventional culture methods. We thus questioned whether three sputa were still necessary to definitively diagnose pulmonary tuberculosis if the BACTEC radiometric culture system were in use. We performed a retrospective analysis of 430 sequential respiratory specimens submitted from 143 patients and from which M. tuberculosis had been recovered by in vitro culture and simultaneously assessed the diagnostic yield of acid-fast smear in this same cohort. M. tuberculosis was recovered from the first specimen for 117 (82%) of the 143 patients, from the second for 14 patients (10%; cumulative rate, 92%), and from the third for 12 patients (8%; cumulative rate, 100%). With the exception of those for bronchial brushings, recovery rates of M. tuberculosis were comparable for all respiratory specimen types (expectorated sputum, induced sputum, tracheal aspirates, bronchoalveolar lavage fluids). Only 46 (32%) of these 143 patients had acid-fast bacilli detected in smears; acid-fast bacilli were detected in the first submitted specimen for 44 patients (96%) and in the second for the remaining 2 patients (4%; cumulative rate, 100%). Culture- or smear-positive rates for sequential specimens obtained from AIDS patients were comparable to those for non-AIDS patients. Overall, the diagnostic culture yield of sequentially submitted specimens was not different from previously published studies in which the BACTEC radiometric culture system had not been used. Despite the documented enhanced ability of the BACTEC 460 radiometric mycobacterial culture system to recover M. tuberculosis more often and faster than conventional methods, three sequential respiratory specimens (regardless of type) were still necessary to definitively diagnose pulmonary tuberculosis.

Acquired Immunodeficiency Syndrome↗
PubMed2000

Identification and detection of Stenotrophomonas maltophilia by rRNA-directed PCR.

Stenotrophomonas maltophilia has recently emerged as an important nosocomial pathogen in immunocompromised patients, in transplant recipients, and in persons with cystic fibrosis (CF). While this organism is nonpathogenic in healthy individuals, it is increasingly associated with morbidity and mortality in susceptible populations. Recent studies have indicated that for approximately 10% of CF patients with moderate lung disease, S. maltophilia can be cultured from respiratory tract secretions. Identification of S. maltophilia can be problematic, and analysis of isolates from the Burkholderia cepacia Research Laboratory and Repository showed that several isolates presumptively identified as B. cepacia by clinical microbiology laboratories were in fact S. maltophilia. To overcome the problems associated with definitive identification, we developed species-specific PCR (SS-PCR) primers, designated SM1 and SM4, directed to the 23S rRNA gene, and tested their utility to accurately identify S. maltophilia directly from sputum. The SS-PCR was developed and tested against a panel of 112 S. maltophilia isolates collected from diverse geographic locations. To test for specificity, 43 isolates from 17 different species were analyzed. PCR with the SM1-SM4 primer pair and isolated genomic DNA as a template resulted in amplification of a band from all S. maltophilia isolates and was uniformly negative for all other species tested, yielding a sensitivity and a specificity of 100% for the SS-PCR. The utility of the SS-PCR to directly identify S. maltophilia in sputum was examined. Thirteen expectorated sputum samples from CF patients were analyzed by SS-PCR. Three samples were PCR positive, in complete concordance with the conventional laboratory culture. Thus, we have developed an SS-PCR protocol that can rapidly and accurately identify S. maltophilia isolates and which can be used for the direct detection of this organism in CF patient sputum.

Cloning, Molecular↗
PubMed1977

Gram stain evaluation of the quality of sputum specimens for mycobacterial culture.

A group of 34 mycobacteria, consisting of 25 Mycobacterium tuberculosis and nine strains of three other species, was isolated from 400 expectorated sputum specimens submitted on 148 patients from county-wide sources. Eight strains (24% of the total) were isolated from specimens evaluated by Gram stain to be oropharyngeal fluids. The remaining 26 strains were isolated from ungradable specimens and those primarily of lower respiratory origin. It was concluded that the random examination of sputum by Gram stain to determine the specimen's quality for mycobacterial isolation is not necessary.

Humans↗
PubMed1978

Diagnosis of pneumococcal pneumonia by antigen detection in sputum.

Pneumococcal polysaccharide was detected by counterimmunoelectrophoresis in the sputum of 20 of 26 (77%) adults with community-acquired pneumonia and a positive sputum culture for Streptococcus pneumoniae. The test was negative in 29 pneumonia patients with negative sputum culture for S. pneumoniae. Pneumococcal antigen was also detected in the sputum of six of nine adults with chronic bronchitis and a positive sputum culture, but was not detected in expectorated respiratory secretions of 22 pneumococcal carriers with colds. Pneumococcal antigen could also be detected in sputum by immunodiffusion; antigen titers varied from 1:2 to 1:256. These results strongly suggest that the detection of pneumococcal antigen in respiratory tract secretions indicates infection caused by S. pneumoniae.

Adult↗
PubMed2004

Intravenous immunoglobulin for cystic fibrosis lung disease: a case series of 16 children.

BACKGROUND AND OBJECTIVE: Some children with severe cystic fibrosis (CF) lung disease develop chest tightness, recurrent dry cough, and intractable wheeze, often accompanied by deteriorating lung function and failure to expectorate sputum. In an attempt to reduce the use of regular oral corticosteroids, we treated a group of such children with monthly courses of intravenous immunoglobulin (IVIG). METHODS: This is a retrospective case note review of 16 children, aged 3-16 years (median 13.0 years) who received 1-66 (median 7.5) courses of monthly IVIG, at a dose of 1 g/kg on two successive days for the first dose, followed by 1 g/kg monthly as a 12 hour infusion, with corticosteroid and antihistamine cover. RESULTS: FEV1 improved from a median (95% confidence interval (CI)) of 50% (39 to 61%) to 54% (48 to 66%), with a median (95% CI) difference of +7.5% (-1.5 to 14.5%; NS). FVC improved from 65% (60 to 77%) to 83% (70 to 89%), with a difference of +13% (4 to 22%, p = 0.01). The total daily dose/kg body weight of oral prednisolone was reduced from 0.6 (0.3 to 1.0) to 0 (0 to 0.1) mg/kg/day, with a reduction of -0.6 (-1.0 to -0.1, p = 0.006) mg/kg/day. The total daily dose of inhaled corticosteroid (budesonide equivalent) was a median (range) of 2000 microg (800-6000 microg), which was reduced to 1500 microg (0-3200 microg). The median (95% CI) difference was -400 microg (-1600 to 0 microg), p<0.05. IVIG was well tolerated and the regimen acceptable to all but one of the children. The following transient adverse reactions were seen in only one patient each: headache, fever, hypotension, aseptic meningitis, and chest tightness. CONCLUSION: We suggest that an n = 1 trial of IVIG in carefully selected patients with severe obstructive CF lung disease is worth considering, as for some it may lead to significant benefit.

Adolescent↗
PubMed2006

Nasopharyngeal aspiration for diagnosis of pulmonary tuberculosis.

BACKGROUND: Confirmation of pulmonary tuberculosis (PTB) in young children is difficult as they seldom expectorate sputum. AIM: To compare sputa obtained by nasopharyngeal aspiration and by sputum induction for staining and culture of Mycobacterium tuberculosis. PATIENTS AND METHODS: Patients from Mulago Hospital, Kampala with symptoms suggestive of PTB were considered for inclusion in the study. Those with a positive tuberculin test and/or a chest radiograph compatible with tuberculosis were recruited. Infection with human immunodeficiency virus (HIV) was confirmed by duplicate enzyme-labelled immunosorbent assay or in children <15 months by polymerase chain reaction (PCR). Direct PCR was undertaken on 82 nasopharyngeal aspirates. RESULTS: Of 438 patients referred, 94 were recruited over a period of 5 months. Median (range) age was 48 (4-144) months. Of 63 patients tested, 69.8% were infected with HIV. Paired and uncontaminated culture results were available for 88 patients and smear results for 94 patients. Nasopharyngeal aspirates were smear-positive in 8.5% and culture-positive in 23.9%. Induced sputa were smear-positive in 9.6% and culture positive in 21.6%. Overall, 10.6% were smear-positive, 25.5% were culture-positive and 26.6% had smear and/or culture confirmed tuberculosis. Direct PCR on nasopharyngeal aspirates had a sensitivity of 62% and specificity of 98% for confirmation of culture-positive tuberculosis. CONCLUSIONS: Nasopharyngeal aspiration is a useful, safe and low-technology method for confirmation of PTB and, like sputum induction, can be undertaken in outpatient clinics.

Child↗