Identification of a mutation associated with erythromycin resistance in Bordetella pertussis: implications for surveillance of antimicrobial resistance.

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.

Laboratory diagnosis of community-acquired lower respiratory tract infection.

This article has focused on the evaluation of outpatients with lower respiratory illness. In large part, the need for microbiological work-up is host-dependent. Healthy patients usually do well, and laboratory data are often unnecessary. The abnormal host requires a different approach and, in general, the more compromised the host, the more aggressive the laboratory evaluation. A renal transplant patient with respiratory symptoms often follows the dictum that "common things happen commonly;" however, the clinician needs that extra level of assurance in this case. Some transplant patients may have respiratory illness caused by strongyloidiasis. Cystic fibrosis is another example of the need for a more comprehensive laboratory evaluation. Specialized selective media and additional susceptibility studies may be needed to evaluate isolates associated with exacerbation of symptoms in these patients. The clinical laboratory should be forewarned of any materials coming from invasive diagnostic techniques, so they can prepare and offer useful advice regarding specimens, transport, and follow-up. Microbiological laboratories are often most knowledgeable regarding what type of testing is appropriate. Direct communication with the laboratory is essential to assure the best patient care.

Fungal pneumonias.

Hundreds of fungal species have been associated with pulmonary diseases in humans, but few are consistently found to cause pneumonia. This paper reviews the clinical presentations, geographic prevalence, and epidemiology of, as well as the most recent, yet readily available, diagnostic methods and general therapy for the more common fungal pneumonias. The chapter is divided into categories based on the fungi's physiological and growth characteristics, clinical presentations, and prevalence in the environment. Sections include the geographically restricted dimorphic fungi (e.g., Blastomyces, Coccidioides, Histoplasma, Paracoccidioides, Sporotrichum, and Penicillium mameffei), the more globally found yeasts (e.g., Cryptococcus neoformans and Candida), other hyaline moulds (e.g., Aspergillus, Zygomyces, Fusarium, and Trichosporon), as well as the dematiaceous fungi (e.g., Altemaria, Bipolaris, Curvularia, Pseudallescheria, and Xylohypha). Pneumocystis carinii is also discussed since it is now accepted as being more closely related to the fungi based on analysis of its 16S-like RNA sequences.

Multicenter laboratory evaluation of the bioMérieux Vitek antimicrobial susceptibility testing system with 11 antimicrobial agents versus members of the family Enterobacteriaceae and Pseudomonas aeruginosa.

A four-center study in which a total of 1,082 recent clinical isolates of members of the family Enterobacteriaceae and Pseudomonas aeruginosa were examined versus 11 antimicrobial agents with the bioMérieux Vitek susceptibility test system (Hazelwood, Mo.) and the GNS-F6 card was conducted. In addition, a challenge set consisting of the same 200 organisms was examined in each of the four participating laboratories. Results obtained with the Vitek system were compared to MICs determined by a standardized broth microdilution method. For purposes of comparison, susceptibility categories (susceptible, intermediate, or resistant) were assigned on the basis of the results of both methods. The result of the broth microdilution test was considered definitive. The total category error rate with the Vitek system and the recent clinical isolates (11,902 organism-antimicrobial comparisons) was 4.5%, i.e., 1.7% very major errors, 0.9% major errors, and 1.9% minor errors. The total category error rate calculated from tests performed with the challenge set (i.e., 8,800 organism-antimicrobial comparisons) was 5.9%, i.e., 2.2% very major errors, 1.1% major errors, and 2.6% minor errors. Very major error rates higher than the totals were noted with Enterobacter cloacae versus ampicillin-sulbactam, aztreonam, ticarcillin, and ticarcillin-clavulanate and with P. aeruginosa versus mezlocillin, ticarcillin, and ticarcillin-clavulanate. Major error rates higher than the averages were observed with Proteus mirabilis versus imipenem and with Klebsiella pneumoniae versus ofloxacin. Excellent overall interlaboratory reproducibility was observed with the Vitek system. The importance of inoculum size as a primary determinant in the accuracy of susceptibility test results with the Vitek system was clearly demonstrated in this study. Specifically, when an inoculum density fourfold higher than that recommended by the manufacturer was used, high rates of false resistance results were obtained with cell wall-active antimicrobial agents versus both the Enterobacteriaceae and P. aeruginosa.

Disseminated and cerebral infection due to Nocardia farcinica: diagnosis by blood culture and cure with antibiotics alone.

Systemic infections with Nocardia species continue to be a serious threat to immunosuppressed hosts. Diagnosis of these infections can be difficult despite their known tendency for cerebral and subcutaneous involvement. We describe a patient who presented with nonspecific constitutional symptoms and was found to have subcutaneous and cerebral abscesses due to Nocardia farcinica. In addition, a blood culture yielded the organism. The patient responded remarkably to oral therapy; resolution of the cerebral disease was observed on serial magnetic resonance images. We discuss the important clinical features on N. farcinica infection, the rarity of positive blood cultures, and the importance of susceptibility testing of Nocardia species in selecting drug therapy.

Mycobacterium haemophilum: microbiology and expanding clinical and geographic spectra of disease in humans.

Reports of the association of Mycobacterium haemophilum with disease in humans have greatly increased. At least 64 cases have now been reported, with symptoms ranging from focal lesions to widespread, systemic disease. The organism is now known to cause primarily cutaneous and subcutaneous infection, septic arthritis, osteomyelitis, and pneumonitis in patients who are immunologically compromised and lymphadenitis in apparently immunocompetent children. Underlying conditions in the compromised patients have included AIDS; renal, bone marrow, and cardiac transplantation; lymphoma; rheumatoid arthritis; marrow hypoplasia; and Crohn's disease. Reports have originated from diverse geographic areas worldwide. The epidemiology of M. haemophilum remains poorly defined; there appears to be a genetic diversity between strains isolated from different regions. The organism is probably present in the environment, but recovery by sampling has not been successful. M. haemophilum has several unique traits, including predilection for lower temperatures (30 to 32 degrees C) and requirement for iron supplementation (ferric ammonium citrate or hemin). These may in the past have compromised recovery in the laboratory. Therapy has not been well elucidated, and the outcome appears to be influenced by the patient's underlying immunosuppression. The organisms are most susceptible to ciprofloxacin, clarithromycin, rifabutin, and rifampin. Timely diagnosis and therapy require communication between clinician and the laboratory.

Primary amoebic meningoencephalitis with Naegleria fowleri: clinical review.

Two children with primary amoebic meningoencephalitis secondary to Naegleria fowleri are reported. Both children died, and the causative agent was identified at autopsy. Presentation and outcome conformed to the usual course of primary amoebic meningoencephalitis and reaffirm the gravity and rapid progression of this infection. The epidemiology, microbiology, diagnostic considerations, and treatment are discussed. Primary amoebic meningoencephalitis should be considered in the differential diagnosis of children with meningitis or encephalitis.

Evaluation of Etest for susceptibility testing of rapidly growing mycobacteria.

MICs of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, and imipenem were determined by Etest for 100 clinical strains of rapidly growing mycobacteria and compared with MICs determined by a reference agar dilution method. Etest MICs were also determined by an alternative inoculum application (agar overlay) method and compared with MICs determined by the inoculum application method recommended by the manufacturer (swabbing). Agreement between Etest and agar dilution MICs within +/- 1 log2 dilution was 85% (511 of 600), and agreement within +/- 2 log2 dilutions was 97% (580 of 600). The rate of complete category agreement was 88%, and rates of major and minor errors were 2.2 and 11.7%, respectively. No very major errors were detected for Etest MICs. Interlaboratory agreement between MICs determined at two separate laboratories was 81% (121 of 149) within +/- 1 log2 dilution and 92% (137 of 149) within +/- 2 log2 dilutions. Agreement between laboratories by interpretive category was 92%. Exact agreement between agar overlay and swab application MICs was 52.3%, and agreement within +/- 1 log2 dilution was 82.3%. Diffuse ellipse edges and trailing growth were still a problem with the overlay method, and in some cases results were more difficult to interpret than they were with the corresponding swab-prepared plate. In summary, our data suggest that Etest may be an accurate and reproducible method for determining susceptibility of rapidly growing mycobacteria.

Bacillus licheniformis bacteremia: five cases associated with indwelling central venous catheters.

Bacillus species are being more frequently recognized as pathogens in immunocompromised hosts or in patients with cancer and central venous catheters. Only nine cases of Bacillus licheniformis infection have been reported in the English-language literature since 1966. In a retrospective study we describe six patients and 17 episodes of B. licheniformis bacteremia over a 5-year span. All six patients had either a Hickman or a Broviac catheter in place for more than 3 months. Five of the six patients had multiple clinically significant episodes of bacteremia due to B. licheniformis. The six patients ranged in age from 4 years to 62 years. Two patients had leukemia or lymphoma and three patients had solid tumors, but only one patient was neutropenic. No deaths were related to B. licheniformis bacteremia. B. licheniformis should be considered as a potential pathogen in immunocompromised patients, especially when bacteremia is associated with the presence of long-term central venous catheters. Mortality due to B. licheniformis bacteremia is low, but recurrent bacteremia due to this organism causes significant morbidity and usually necessitates removal of the catheter.

Comparative evaluation of the E test for susceptibility testing of Nocardia species.

The E Test (PDM Epsilometer; AB Biodisk, Solna, Sweden) was compared with microbroth dilution and disk diffusion for antimicrobial susceptibility testing of Nocardia as a collaborative study by two geographically separate laboratories. A total of 52 clinical isolates and five species of Nocardia were used in this comparative evaluation. Susceptibility testing was performed with Mueller-Hinton media and eight antimicrobial agents. Growth of the test strains with Mueller-Hinton medium was generally satisfactory, with the majority of isolates producing adequate growth within 24-36 h. Growth inhibition ellipses were generally well delineated and uniform for most drugs, and the points of intersection with the E Test strip were generally easy to determine. An inoculum size of approximately 2.0 x 10(7) CFU/ml was optimal for performance of the E Test method with the Nocardiae. Comparison of E Test and microbroth dilution MICs revealed 89.4% agreement for all drugs within +/- 1 log2 dilution. Using NCCLS interpretive criteria for susceptible and resistant results, complete agreement between E Test and disk diffusion results was 93.3%, and between E Test and microbroth dilution results was 96.2%. Interpretive category errors occurred at rates of 18.2% (risk corrected), 0, and 4.1% for very major, major, and minor errors, respectively, when E Test results were compared with disk diffusion results, and 0, 0, and 3.8%, respectively, when E Test was compared with microbroth dilution. Inter- and intra-laboratory reproducibility, within +/- 1 log2 dilution for all drugs, was 95% and 98%, respectively. Results from this study suggest that E Test may be suited for use as an alternative method for antimicrobial susceptibility testing of Nocardia species.

Comparison of BACTEC Plus 26 and 27 media with and without fastidious organism supplement with conventional methods for culture of sterile body fluids.

We compared the BACTEC Plus 26/27 culture system (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) with and without fastidious organism supplement with conventional centrifugation preparation and plating for the recovery and speed of detection of microorganisms. A total of 1,101 sterile body fluid specimens were collected and processed at five hospital laboratories, yielding 234 (21%) positive cultures. Of the 176 isolates considered clinically significant, 133 (76%) were recovered by both the BACTEC system and conventional culture, while 28 (16% [P < 0.005]) were recovered by BACTEC only and 11 (6%) were recovered by conventional culture alone. There were no statistically significant differences in the speed of detection of microbial growth. It was found that BACTEC, with or without the addition of fastidious organism supplement, exhibited improved sensitivity for the recovery of microorganisms, including fastidious bacteria.

Comparison of two commercial broth-culture systems for microbial detection in dialysates of patients on continuous ambulatory peritoneal dialysis.

The capability of two commercial systems was studied for microbial detection in dialysis effluents from patients on continuous ambulatory peritoneal dialysis (CAPD). Two methods of processing dialysate from patients on CAPD were compared: (a) direct inoculation of 10 ml of dialysate into a single bottle broth culture system (Signal; Oxoid, U.S.A., Columbia, Maryland) and 5 ml into each of two BACTEC blood culture bottles (Johnston Laboratories, Towson, Maryland); and (b) centrifugation of 50 ml of dialysate and culture of the sediment in an Oxoid bottle. Of the 196 specimens cultured, 99 (51%) yielded growth. Recovery rates of significant isolates were 95% for the BACTEC system, 89% for the Oxoid system, and 88% for the centrifugation-Oxoid system. Recovery of eight isolates from the Oxoid system was by subculture rather than a visual "signal." The Oxoid system is a practical, cost-effective, alternative culture method for effluents from CAPD patients in laboratories not having access to the BACTEC system.

Use of the Limulus amebocyte lysate test as a cost-effective screen for gram-negative agents of meningitis.

A retrospective review of data from two institutions demonstrated that the Limulus amebocyte lysate test was a simple and cost-effective means to screen cerebrospinal fluids (CSF) for Gram-negative agents of meningitis. Results of either gelation Limulus amebocyte lysate (LAL) or chromogenic Limulus amebocyte lysate (CLAL) tests on 1504 CSF were evaluated along with results of antigen detection tests [ADT; either latex agglutination (LA) or counterimmunoelectrophoresis (CIE)], cultures, and Gram-stains. All 127 CSF positive for Haemophilus or Neisseria by culture and/or ADT were also positive by Limulus test (100%). The sensitivities of Gram stain examination, culture, LA, and CIE for these two pathogens were 81%, 91%, 86%, and 63%, respectively. The Limulus test on lumbar CSF was positive in 67% of cases with other Gram-negative bacillary meningitis, in 33% of cases with Gram-negative bacillary abscess or ventriculitis, in none of the cases with Gram-positive, mycobacterial, treponemal, fungal, or viral meningitis, and in 1% of cases with either normal or otherwise negative bacteriologic findings. Overall, sensitivity and specificity of the Limulus test were 97% and 99%, respectively. The cost of either version of the Limulus test was less than 15% of the cost of ADT batteries for Haemophilus and Neisseria. Based on its sensitivity for Haemophilus and Neisseria in CSF, we propose its use as a cost-effective screen to minimize the need for the more expensive ADT batteries.

Phaeohyphomycosis caused by the fungal genera Bipolaris and Exserohilum. A report of 9 cases and review of the literature.

We have reported 7 new cases of Bipolaris infection and 2 of Exserohilum infection, which demonstrate the capability of these 2 genera to cause invasive as well as "allergic" disease. As noted previously, it is likely that all of the cases of "Helminthosporium" and Drechslera infections reported in the literature were caused by Bipolaris or Exserohilum. Infections due to these 2 genera are probably more common than previously recognized. They should be included in the differential diagnosis of central nervous system and disseminated fungal disease, sinusitis, keratitis, peritonitis associated with continuous ambulatory peritoneal dialysis, and allergic bronchopulmonary disease. These various entities have distinct histopathologic characteristics. With disseminated disease in the immunocompromised patient, the most frequent findings are acute inflammation with prominent vascular invasion, thrombosis, and infarction. In contrast, granulomatous inflammation and leukocytoclastic vasculitis are seen in meningoencephalitis caused by these fungi. The histologic features of allergic bronchopulmonary disease and sinusitis are similar. A chronic inflammatory infiltrate of lymphocytes, plasma cells and eosinophils within edematous granulation tissue is found in addition to squamous metaplasia and thickening of the basement membrane. Infections caused by Bipolaris/Exserohilum and Aspergillus show many clinical and pathologic similarities despite the lack of taxonomic relationship between these fungi. Both cause disseminated disease in immunocompromised patients that is characterized by tissue necrosis and vascular invasion. Both cause central nervous system disease, osteomyelitis, and sinusitis and are associated with allergic bronchopulmonary disease. Sinusitis, the most common form of disease caused by Bipolaris and Exserohilum, occurs in otherwise healthy patients with nasal polyposis and allergic rhinitis. Although pathologic evidence of bone invasion may not be found, there frequently is radiographic evidence of invasive disease. Most patients who are treated initially with surgical debridement and amphotericin B have apparently been cured. However, longer follow-up will be necessary in these patients. Amphotericin B appears to be the treatment of choice for invasive infections caused by Bipolaris/Exserohilum species. Ketoconazole and other imidazole derivatives may also be effective in certain of the disease entities caused by these black moulds; however, their role has yet to be defined.(ABSTRACT TRUNCATED AT 400 WORDS)

Screening for bacteriuria with the LN strip test.

Recently, the LN strip test was introduced for purposes of rapid screening of urine specimens for bacteriuria. The LN strip test permits detection of urinary leukocyte esterase and nitrite. A total of 2,481 unselected urine specimens from three tertiary care hospitals were examined using the LN strip test and the results were compared with those obtained with a conventional quantitative culture technique. When the results of the leukocyte esterase and nitrite tests were combined, the sensitivity and specificity of the LN strip test were 88.8% and 71.3%, respectively, based on culture results of greater than or equal to 10(5) CFU/ml. Test sensitivity decreased at greater than or equal to 10(4) CFU/ml, whereas test specificity increased.

Experience with needle biopsy for coccidioidal lung nodules.

Three hundred forty-eight patients with solitary pulmonary nodules underwent needle biopsy of the lung in an endemic area for coccidioidomycosis. The purposes of the study were to establish the value of smears and cultures from needle biopsy specimens in the diagnosis of solitary coccidioidal granuloma and to determine how the final diagnosis was established for patients without a clear-cut diagnosis after initial biopsy. Spherules of Coccidioides immitis were initially identified in 49 of the 96 patients with coccidioidomas. Only three of 35 cultures from biopsy-proved coccidioidal lesions were positive (8.6% yield); nevertheless, cultures were cost-effective in nondiagnostic cases. Thoracotomy should be delayed until the results of cultures are known if findings of skin tests, serologic studies, and roentgenography make a benign diagnosis plausible.