Lung transplantation for cystic fibrosis patients with Burkholderia cepacia complex. Survival linked to genomovar type.

The number of cystic fibrosis (CF) patients undergoing lung transplant has risen over the past decade, because of a clear-cut survival benefit. However, patients with Burkholderia cepacia complex are often excluded from transplantation because of increased mortality. To determine the influence of B. cepacia complex genomovar type on transplant outcome, we undertook a retrospective study in 121 CF patients transplanted at UNC. Twenty-one and three patients, respectively, were infected pre- or postoperatively with B. cepacia complex. All posttransplant acquisitions were successfully treated. However, excess mortality occurred over the first 6 postoperative months in those infected preoperatively with B. cepacia complex compared with those not infected (33% versus 12%, p = 0.01). The 1-, 3-, and 5-yr survival were significantly lower in the B. cepacia complex cohort. Of the patients infected preoperatively, genomovar III patients were at the highest risk of B. cepacia complex-related mortality (5 of 12 versus 0 of 8, one isolate not typed; p = 0.035). Each of the B. cepacia complex-related deaths was caused by a unique genotype as determined by pulsed-field gel electrophoresis. All isolates were negative for the cable pilin gene. These results warrant a multicenter analysis of B. cepacia complex-infected patients with genomovar-typing to confirm that genomovar III patients are at highest risk for post-transplant complications.

Utility of obtaining blood cultures in febrile neutropenic patients undergoing bone marrow transplantation.

Infection remains an important cause of morbidity and mortality after bone marrow or stem cell transplantation. To evaluate the role of obtaining blood cultures for intermittent or persistent fever in neutropenic patients on antibiotic therapy, we performed a retrospective chart review of 196 consecutive patients admitted to the Bone Marrow Transplant Unit at the University of North Carolina Hospitals from 1995 to 1998. From the cohort of 196 patients, 154 patients developed neutropenic fever. The initial blood culture was positive in 16 of 145 patients during the first fever episode giving a prevalence of 11%. From the total of 109 patients that had blood cultures drawn after day 1 of fever, five patients had blood cultures positive for a pathogen, a prevalence of 4.6%. In only one patient, did blood cultures drawn after day 1 identify an organism not present on day 1 (prevalence 0.9%). After reviewing the results in the first 105 patients, we changed our timing of collection of blood cultures. Forty-nine patients were treated in this manner and we found that the mean number of blood cultures decreased from 9.2 to 4.7 per patient without a change in the frequency of infectious complications or length of hospitalization.

Escherichia coli. EAEC, EHEC, EIEC, ETEC.

There are at least four different ways in which E. coli can cause diarrheal disease: invasion of the intestinal epithelium, enterotoxin production, STx production, and adherence with disruption of the normal functioning of the intestinal epithelium. Fecally contaminated food and water are the source of E. coli infections in humans. Travelers from industrialized countries with good sanitation systems are at risk for obtaining these organisms when they travel to the developing world, where these organisms are endemic. In the developing world, these organisms are a major cause of infant mortality. Infections in adults are usually self-limited and typically respond to oral rehydration therapy. Only in severe illness is antimicrobial therapy needed. Prevention of these infections requires good sanitation and food handling practices. In addition, travelers to the developing world should avoid certain types of food and contaminated water.

Invasive group A streptococcal infections in North Carolina: epidemiology, clinical features, and genetic and serotype analysis of causative organisms.

During 1994 and 1995, an increase in the number and severity of group A streptococcal (GAS) infections was noted in North Carolina. Ninety-six patients had GAS recovered from blood and other sterile body fluids, abscesses, and soft tissue. The overall case fatality rate was 11% but was much higher in patients with toxic shock syndrome (55%) and necrotizing fasciitis (58%). Recent invasive GAS isolates were compared with pre-1994 invasive isolates and temporally related pharyngeal isolates by M protein serotyping, pulsed field gel electrophoresis (PFGE), and polymerase chain reaction amplification of the streptococcal pyrogenic exotoxin A gene. Serotypes M1 and M3 accounted for 50% of recent invasive isolates (1994-1995) and 58% of pharyngeal isolates (1994). The latter isolates demonstrated PFGE patterns that were identical to invasive M1 and M3 strains, suggesting that pharyngeal infections may have served as a reservoir for virulent GAS clones.

The effects of panresistant bacteria in cystic fibrosis patients on lung transplant outcome.

The number of cystic fibrosis (CF) patients undergoing lung transplant continues to rise as long term survival improves. One major contraindication to this potentially life-saving intervention is infection with multi-drug-resistant bacteria. We undertook this retrospective study in 66 transplanted patients over 6 yr to determine the influence of panresistant bacteria on transplant outcome. The in vitro antibiotic susceptibility pattern of respiratory tract bacteria obtained pre- and/or intraoperatively was used to categorize patients into panresistant (n = 27) (Burkholderia cepacia, n = 6, and Pseudomonas aeruginosa, n = 21) or sensitive (n = 39) groups. Postoperative ventilator days, hospital length of stay, and antibiotic days were similar for both groups (p > 0.2). The incidence of bacterial bronchitis (28% and 33%, respectively) and pneumonia (28% and 38%, respectively) did not differ between these groups (p > 0.2) at 6 mo. Likewise, one-year (81% and 83%, respectively) survival was similar for both groups (p > 0.2). As expected, panresistant B. cepacia patients had a lower 1-yr survival (50% versus 90%, p < 0.05) and had a higher mortality attributable to B. cepacia (50% versus 0%, p < 0.01) compared with panresistant P. aeruginosa patients. Our results indicate that CF patients infected with panresistant P. aeruginosa have similar transplant outcomes as patients with sensitive bacteria and should not be excluded from lung transplant based solely on this criterion.

Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination.

A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens. Methods included direct assay of cytotoxin in stool by tissue culture, C. difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C. difficile test (Meridian Diagnostics, Inc.). The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C. difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes). Evaluation for C. difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C. difficile in stool specimens. The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C. difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods). The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C. difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%). The data support the use of the ImmunoCard C. difficile test as an adjunct for the diagnosis of CDAD.

Accuracy of four commercial systems for identification of Burkholderia cepacia and other gram-negative nonfermenting bacilli recovered from patients with cystic fibrosis.

Burkholderia cepacia has recently been recognized as an important pathogen in chronic lung disease in patients with cystic fibrosis (CF). Because of the social, psychological, and medical implications of the isolation of B. cepacia from CF patients, accurate identification of this organism is essential. We compared the accuracies of four commercial systems developed for the identification of nonfermenting, gram-negative bacilli with that of conventional biochemical testing for 150 nonfermenters including 58 isolates of B. cepacia recovered from respiratory secretions from CF patients. The accuracies of the four systems for identifying all nonfermenters ranged from 57 to 80%, with the RapID NF Plus system being most accurate. The accuracies of these systems for identifying B. cepacia ranged from 43 to 86%, with the Remel system being most accurate. Depending on the commercial system, from two to seven isolates were misidentified as B. cepacia. The relatively poor performance of the commercial systems requires that identification of certain nonfermenters be confirmed by conventional biochemical testing. These organisms include B. cepacia, Burkholderia sp. other than B. cepacia, and infrequently encountered environmental species (Pseudomonas and Flavobacterium species). In addition, conventional biochemical testing should be done if a commercial system fails to assign an identification to an organism. Confirmatory testing should preferably be performed by a reference laboratory with experience in working organisms isolated from CF patients.

Quality assurance study of bacterial antigen testing of cerebrospinal fluid.

Bacterial antigen testing (BAT) of cerebrospinal fluid (CSF) by latex agglutination is a low-yield procedure in patients whose CSF specimens have normal laboratory parameters. Between August 1992 and August 1994, we evaluated 287 bacterial antigen (BA) test requests to determine whether yields could be improved and whether patient costs could be reduced by cancelling BAT for those patients with normal CSF parameters (cell count, protein, glucose) after consultation with physicians. A total of 171 (68%) BA tests were canceled by this approach. None of these CSF specimens was culture positive for an organism detectable by BAT. Of the remaining 116 CSF specimens tested, only 3 were positive by BAT, one each for Neisseria meningitidis, Streptococcus pneumoniae, and group B streptococcus. Only 43 of the CSF specimens tested had at least two abnormal parameters; the 3 positive CSF specimens were included in this group. In light of the low rate of positivity, the number of BA tests can be further reduced by establishing criteria that must be met before a CSF specimen is accepted for BAT. After review of our data and the literature concerning this topic, we concluded that only specimens with leukocyte counts of > or = 50 cells per mm3 should be tested. Of 287 specimens evaluated in our study, only 36 met this criterion, including the 3 BA-positive specimens. Enacting such a restriction would have reduced the total number of BA tests by 251 (87%) without compromising patient care. A laboratory cost savings of $6,500 per year would have been realized, with a substantial reduction in the cost per positive test. Patient charges would have been reduced by $12,500 per year.

Transmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence suggesting capsular transformation of the resistant strain in vivo.

Surveillance for nasopharyngeal colonization with Streptococcus pneumoniae was maintained in a research day care center between 1985 and 1992. An outbreak of nasal carriage of a multi-drug-resistant (MDR) serotype 23F organism occurred between May 1990 and December 1991 involving 14 of 52 children. Electrophoresis of penicillin-binding proteins (PBP) and pulsed-field gel electrophoresis (PFGE) of chromosomal DNA indicated that the MDR serotype 23F organism was closely related to a serotype 23F MDR clone that has been prevalent in Spain since the early 1980s. In June 1991, an MDR serotype 14 organism was isolated from a child who had previously carried the MDR serotype 23F strain. PFGE and PBP typing revealed that the MDR serotype 14 organism was very similar to the circulating MDR serotype 23F strain, suggesting serotype transformation. Dissemination of MDR pneumococcal strains and possibly spread of the MDR phenotype to additional serotypes may be facilitated in group day care.

In vivo and in vitro studies of Clostridium difficile-induced disease in hamsters fed an atherogenic, high-fat diet.

After previous observation of increased susceptibility to Clostridium difficile enterocolitis in hamsters fed an atherogenic, high-fat diet, a study was undertaken to examine experimental reproducibility of this disease. Hamsters were fed either the high-fat diet or a control diet, then orally challenged with a toxigenic strain of C. difficile. Hamsters fed the high-fat diet suffered 80% morbidity, which was statistically significant from the 11% morbidity of the control diet group (P < or = 0.05). The disease presented acutely, the most common presentation being sudden death. The most common lesions in the affected hamsters were necrohemorrhagic cecitis and cecal mucosal hyperplasia. Hepatic lipidosis was consistent in all hamsters fed the high-fat diet. Toxigenic C. difficile could be recovered from the cecum of most affected hamsters, and toxins A and B were detected in these ceca. Hamsters that were fed the high-fat diet and orally inoculated with a nontoxigenic strain of C. difficile before experimental challenge with a toxigenic strain were initially protected against disease. The protection decreased with each exposure to the toxigenic strain. Results of in vitro colonization-resistance studies indicated that the cecal flora from hamsters fed the high-fat diet and control diets inhibited C. difficile growth, suggesting that increased disease susceptibility was not the result of altered cecal flora.

Comparison of antimicrobial susceptibility methods for detection of penicillin-resistant Streptococcus pneumoniae.

We sought to determine if commercially available susceptibility tests were accurate in detecting penicillin resistance and relative resistance in Streptococcus pneumoniae. We compared the reference MIC method with oxacillin disk screening and three commercial tests, E-test (AB Biodisk), JustOne (Radiometer America), and MicroScan Pos MIC Panel Type 6 (Baxter Diagnostics), with 80 selected clinical isolates. Thirty-three additional isolates were tested by the reference method and the E-test to further validate the latter method. Oxacillin screening was effective in detecting all penicillin-resistant and relatively resistant strains of S. pneumoniae. The MicroScan method was not effective in detecting penicillin resistance or relative resistance. The JustOne system classified only 6 (35%) of 17 resistant strains correctly, with 11 resistant strains classified as relatively resistant. The E-test correctly classified 30 (83%) of 36 resistant isolates, with 6 resistant isolates interpreted as relatively resistant. For determining penicillin MICs for S. pneumoniae, the E-test was the most accurate of the commercial systems that we studied.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), primarily from human immunodeficiency virus-infected patients, were tested in this study. Sixty-seven specimens (44 serum and 23 CSF samples) were positive for cryptococcal antigen with both tests, and 511 (282 serum and 229 CSF samples) were negative. The two latex reagents agreed for 326 of 327 serum specimens (44 positives and 282 negatives). One serum specimen with a titer of 1:2 was CALAS positive but CRYPTO-LEX negative. The titer correlation coefficient for the two tests was 0.884 when two highly discordant serum specimens were eliminated from analysis of the data. The two latex tests agreed for 252 of 253 CSF specimens (23 positives and 229 negatives). One specimen with a titer of 1:2 was positive with CALAS and negative by CRYPTO-LEX. The correlation coefficient of the two tests for CSF titers was 0.886. The sensitivity and specificity of CRYPTO-LEX were 97 and 100%, respectively, with a 99.6% correlation with CALAS. These data show that the performance of CRYPTO-LEX is comparable to that of CALAS for detection of cryptococcal antigen in serum and CSF.

Inapparent transmission of Pseudomonas (Burkholderia) cepacia among patients with cystic fibrosis.

Pseudomonas cepacia is a significant pathogen in children and young adults with cystic fibrosis, and prevention of its acquisition has become an important goal in patient management. Although it is now clear that this bacterium can be transmitted from person to person, the frequency of this mode of acquisition and the measures required to prevent it are controversial. In this report we describe the use of a novel genotyping method to extend our previous investigation of person to person transmission of P. cepacia among patients with cystic fibrosis attending an educational program. Three (20%) of 15 individuals acquired P. cepacia after contact with a chronically colonized patient. Analysis revealed that the isolates recovered from the three newly colonized patients were the same as that from the index patient. We also demonstrated that pulmonary colonization with P. cepacia may not be detected by currently recommended culture methods for as long as 2 years after acquisition. These data indicate a need to develop more sensitive means of detecting P. cepacia colonization in order better to understand host-pathogen interaction and to optimize preventive strategies.

Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases.

Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.

Increased adherence of Staphylococcus aureus from cystic fibrosis lungs to airway epithelial cells.

Airway colonization by Staphylococcus aureus is a frequent feature of cystic fibrosis (CF). To assess the pathogenesis of selective colonization with this organism, we compared the capacity of S. aureus isolated from the respiratory tract of CF and non-CF patients to adhere to epithelial cells from the upper and lower airways of CF and control subjects. Bacterial adherence to bronchial epithelial cell lines was significantly greater for CF than for non-CF isolates (p < 0.001). Of 17 CF S. aureus isolates 12 adhered at a level > 1 bacterium per cell; this was true for only 1 of 14 non-CF isolates. CF S. aureus isolates also bound more avidly than non-CF isolates to ciliated (p < 0.05) and squamous nasal cells (p < 0.02) and buccal epithelial cells (p < 0.005) freshly harvested by scraping. Each S. aureus isolate bound with equal avidity to epithelial cells from CF patients and healthy individuals. Adherence was not related to sex, age, severity of pulmonary disease, presence of other microorganisms in the airways, or genotype of the CF hosts. Binding of S. aureus was blocked by proteinase treatment of organisms, suggesting that adherence is mediated by one or more peptide adhesins. We propose that the high prevalence of adherent S. aureus is due either to selection of adherent strains by CF airways or to induction of an adherent phenotype by factors residing at the CF airways surface.

The use of a commercially available enzyme immunoassay for the detection of Clostridium difficile toxin A.

A commercially available enzyme immunoassay (EIA) for the detection of Clostridium difficile toxin A (Premier Toxin A EIA, Meridian Diagnostics, Cincinnati, Ohio) was compared with tissue culture cytotoxicity assay, enterotoxigenic culture, and latex agglutination test for the laboratory diagnosis of C difficile-associated disease. When evaluated for detection of C difficile-associated disease using clinical specimens, EIA was the most sensitive (83.1%) and tissue culture cytotoxicity assay was the most specific test with EIA, tissue culture cytotoxicity assay and enterotoxigenic culture having similar correlation values (96.6, 96.1, 94.0%, respectively). The latex agglutination test was not as accurate (89.7% correlation) as the other three tests due mainly to its poor sensitivity (47.9%). The EIA is a rapid, easy-to-use alternative to tissue culture cytotoxicity assay for detection of C difficile-associated disease.

Improved recovery of mycobacteria from respiratory secretions of patients with cystic fibrosis.

Pulmonary colonization and infection of patients with cystic fibrosis by Mycobacterium spp. has recently been recognized as a potentially important clinical problem. However, frequent contamination of mycobacterial cultures by pseudomonads has hampered efforts to define the extent of this problem. This study was done to evaluate current techniques and to establish a more efficient method of recovering mycobacteria from respiratory secretions of patients with cystic fibrosis. Decontamination of respiratory specimens (n = 121) with 0.25% N-acetyl-L-cysteine and 1% sodium hydroxide (NALC-NaOH) was associated with a high rate of pseudomonas overgrowth for both Lowenstein-Jensen slants (74%) and BacTec vials supplemented with PANTA (polymyxin B [50 U/ml], amphotericin B [5 micrograms/ml], nalidixic acid [20 micrograms/ml], trimethoprim [5 micrograms/ml], azlocillin [10 micrograms/ml]) (36%). This overgrowth limited recovery of mycobacteria to only 64% (9 of 14) of specimens positive by smear for acid-fast bacilli (AFB). Decontamination of specimens (n = 441) with NALC-NaOH, followed by 5% oxalic acid treatment, resulted in contamination of only 5% of Lowenstein-Jensen slants and 3% of BacTec vials. AFB were recovered from all 90 AFB smear-positive specimens following the use of this decontamination technique. We recommend that respiratory secretions be decontaminated with NALC-NaOH and oxalic acid to decrease the incidence of Pseudomonas aeruginosa overgrowth.

Pertussis is rare in human immunodeficiency virus disease.

Many adults are susceptible to pertussis, and Bordetella pertussis has been isolated from five patients with HIV disease. The prevalence of B. pertussis in 60 HIV-infected adults with nasopharyngeal (NP) swab cultures were studied and questionnaires were used that assessed HIV-related risk behaviors and disease status, immunization history, and symptoms of respiratory disease. Although 72% had cough and 33% had cough for > 14 days, no nasopharyngeal (NP) swab cultures were positive for Bordetella species. Of the 44 (73%) patients who had follow-up NP swab cultures at 6 months, all were still negative. On the basis of these data from our HIV-infected population, the estimated population prevalence of pertussis is zero, with an upper 95% confidence limit of 0.00065, or fewer than 6.5 cases of pertussis per 10,000 HIV-infected adults. Given this low prevalence, HIV-infected patients with respiratory symptoms do not appear to be a reservoir for B. pertussis in the community.

Nontuberculous mycobacteria in adult patients with cystic fibrosis.

Because patients with cystic fibrosis (CF) may be predisposed to airway infections with unusual microorganisms, we screened the sputum of adult CF patients for mycobacterial organisms. Acid-fast bacilli (AFB) smears and mycobacterial culture were performed on 297 sputum specimens from 87 patients. Cultures for mycobacteria were frequently overgrown with other bacteria; 22.6 percent of cultures were contaminated. Despite this limitation of mycobacterial culture, 17 patients had at least one positive culture for a Mycobacterium other than tuberculosis (MOTT). Eleven patients were positive for Mycobacterium avium-intracellulare (MAI), two for MAI and M chelonei, three for M chelonei, and one for M fortuitum. None was positive for M tuberculosis. Patients with CF with MOTT were similar to patients with CF without MOTT; only a slightly different (older) age distribution was recognized. The clinical significance of MOTT was difficult to determine in any individual patient, but patients with positive AFB smears appeared more likely to suffer pathogenic effects. We conclude that MOTT is frequently recovered from adult CF patients in the southeastern United States. A specific risk factor for colonization and/or pathogenic infection in this patient group was not evident. The general prevalence and clinical pathogenesis in CF patients in the United States remains to be determined.