Clinical microbiology of bacteremia: an overview.

Newer methodologies for detecting bacteria in blood are more sensitive than conventional procedures. The possibility of contamination from a variety of sources is discussed. The problem of interpreting the findings of some of these techniques is forcing the microbiologist and clinician to reevaluate previously held ideas regarding isolates that are considered insignificant. The aggressive use of foreign bodies, whether of short duration such as central venous catheters or of long duration such as prosthetic heart valves, predisposes patients to a wide variety of infectious complications that are often associated with bacteremia. Staphylococcus epidermidis, Corynebacterium species (particularly group JK), Bacillus species, and S. aureus are discussed.

Inhibition by ascorbic acid (vitamin C) of chemical detection of blood in urine.

Review of results of 9,620 consecutive urinalyses revealed 892 urines that contained microscopic evidence of erythrocytes and 98 for which there were discrepancies between microscopic evidence of hematuria (greater than 20 erythrocytes/high-power field) and chemical detection of blood (0 or "1+" when results should be "3+"). Eleven specimens (from ten patients) showed negative results of chemical tests for blood and greater than 40 erythrocytes/high-power field (HPF). Nine of these patients were receiving ascorbic acid supplementation. In a random sample of 20 patients with greater than 40 erythrocytes/HPF and strongly positive tests for blood, none was receiving ascorbic acid, a significant difference by chi-square analysis. In a prospective study, low levels of ascorbic acid inhibited chemical detection of blood. At 25 mg/dl ascorbic acid, 10--20 erythrocytes/HPF could not be detected; at 35 mg/dl ascorbic acid, greater than 20 erythrocytes/HPF were undetectable. For quantitation of the low level at which ascorbic acid inhibits chemical detection of blood, fresh urine specimens should be analyzed. Ascorbic acid is oxidated in vitro to products that partially inhibit detection of blood yet do not assay as ascorbic acid.

Clinically significant differences in antibiograms of morphologic variants of blood culture isolates.

The present environment of cost containment frequently forces laboratories to reassess various strategies for the diagnostic work-up of patient specimens. The necessity for the performance of antimicrobial susceptibility testing on all morphologic variants of blood culture isolates was examined in this study. Over a 4-year period, of 143 such organisms that were identified, 56 (39%) exhibited clinically significant differences in antibiogram profiles. Coagulase-negative Staphylococcus represented the largest proportion of isolates (n = 115), 39% of which demonstrated clinically significant differences in antibiograms. The authors conclude that these results justify the current practice in their laboratory of performing separate antimicrobial susceptibility testing on all morphologic variants of isolates.

Multicategory interpretive reporting of susceptibility testing with selected antimicrobial concentrations. Ten years of laboratory and clinical experience.

For the last 10 years, the NIH Microbiology Laboratory has been using a broth microdilution method to perform antibiotic susceptibility tests. Instead of using a continuous twofold dilution series as we had done for the previous 10 years, in 1978 we introduced a susceptibility panel that utilized a minimal number of selected (discontinuous) antimicrobic concentrations. The concentrations selected were those thought to be the most clinically relevant, based on known pharmacologic properties of each antimicrobic agent, as well as known MIC population distributions that we had acquired on 50,000 organisms in the preceding years. In addition to using selected concentrations, we also added interpretive codes to aid the physician in selecting the best antimicrobic agent to use. We had previously only reported quantitative MIC results without qualitative interpretations. The present interpretive criteria inform the physician not only if the organism is susceptible or resistant but also if intramuscular or intravenous doses are needed, if the organism is susceptible to an antimicrobic agent but only for lower urinary tract infections, if the organism is resistant to penicillin by virtue of penicillinase production, and in the case of streptococci, if streptomycin or gentamicin can be expected to show synergy when combined with a penicillin. The use of clinically relevant selected concentrations combined with clear interpretive criteria has worked well in our hospital setting. Physicians are able to understand and utilize the information effectively and have found almost no need for exact MICs using a twofold dilution series.

Respiratory cryptosporidiosis in a patient with malignant lymphoma. Report of a case and review of the literature.

Respiratory cryptosporidiosis is a rare complication of intestinal infection by cryptosporidia, with only six cases reported (to our knowledge) since its first description in 1983. We report the first case of respiratory cryptosporidiosis recognized at the National Institutes of Health, Bethesda, Md. An antemortem diagnosis was made based on recognition of acid-fast cryptosporidia in an induced sputum specimen obtained from a 64-year-old woman with malignant lymphoma and an associated profound immunodeficiency. Autopsy confirmed the presence of cryptosporidia along the apical aspect of the respiratory epithelium lining the trachea, bronchi, and bronchioles. Cryptosporidia were also identified in the duodenum and gallbladder. Immunohistochemical staining of the paraffin-embedded autopsy lung sections using a monoclonal antibody verified the diagnosis of cryptosporidiosis. Review of our case and the literature suggests that respiratory cryptosporidiosis is characterized by a chronic tracheitis, bronchitis, and bronchiolitis but generally does not cause severe pulmonary dysfunction.

Mathematical analysis of the API enteric 20 profile register using a computer diagnostic model.

Results for 21 biochemical tests using the API-20 Enteric kit were obtained from the manufacturer's files for 27,820 bacterial isolates. These isolates were identified by the API Profile Register and also by a computer diagnostic model which estimates the relative likelihoods of various identifications. The computer confirmed the identification in the API Profile Register for 99.36% of the isolates. The manufacturer has reviewed areas of the API Profile Register questioned by the computer analysis; a number of resulting modifications to the API Profile Register have been incorporated in an update letter. This computer model provides a convenient and powerful way to interpret a large number of test results for bacterial identification. This study also demonstrates the use of a large collection of isolates to refine the data matrix used by the diagnostic model.

Construction of an interpretive pattern directory for the API 10 S kit and analysis of its diagnostic accuracy.

A directory of test patterns and their interpretations has been prepared for identification of Enterobacteriaceae by using the 11-test API 10 S kit. The diagnostic accuracy of the directory and kit were evaluated by using records of test results for 37,476 isolates studied with the 21-test API 20 Enteric kit. Analysis indicates that 96.9% of the isolates would have been correctly identified at the genus level and 95.9% at the species level by using only the subset of tests included in the API 10 S.

Antimicrobial susceptibilities of mycobacteria as determined by differential light scattering and correlation with results from multiple reference laboratories.

The DAWN Model B laser light scattering instrument (Wyatt Technology Corporation, Santa Barbara, Calif.) was evaluated to assess its potential to provide rapid mycobacterial antimicrobial susceptibility test results. For Mycobacterium tuberculosis there was a clear separation between susceptible and resistant results with the isolates tested, and there was excellent correlation with reference laboratory results. For Mycobacterium avium there was no obvious breakpoint between susceptible and resistant results with the isolates tested, and correlation with reference laboratory results was less good than for M. tuberculosis. However, for M. avium there was also less agreement among reference laboratory results than for M. tuberculosis. Significant instrument design and software program changes would be required for the instrument to become a useful tool for mycobacterial susceptibility testing in the diagnostic laboratory.

Quantitation of pathogenic potential of Staphylococcus aureus.

Numerical estimates of the pathogenicity of Staphylococcus aureus strains were made for phage-typed strains from a relative incidence of significant to nonsignificant isolates from hospital patients. For a specific phage-patterned strain, the number of isolates from significant (wounds, abscesses, blood, etc.) sites was divided by the number of isolates from nonsignificant (respiratory tract, body surfaces, etc.) sites. This value, multiplied by 100, was the index of infection potential (IIP). IIP values for the S. aureus strains studied ranged from a low of 8 to a high of 50. The average IIP for all phage-patterned strains that occurred 50 or more times was 20. There was an inverse relationship between length of the phage pattern (number of the 26 typing phages that lysed the strain) and pathogenicity. Those strains with shorter phage patterns had higher IIP values and were more pathogenic. Strains lysed by one phage had an average IIP of 27, whereas those lysed by 18 phages had an average IIP of 14.

Broth dilution minimum inhibitory concentrations: rationale for use of selected antimicrobial concentrations.

A microdilution susceptibility testing procedure utilizing selected, clinically relevant concentrations of a large number of antimicrobial agents is described. A qualitative code designed to facilitate interpretation of quantitative results is coupled with each antimicrobial concentration. Both the antimicrobial concentrations selected for testing and the assigned codes are based on published data regardling attainable antimicrobial levels in serum and urine.

Acquisition of K:1-like antigen during terminal sepsis.

This report describes a patient whose own and transfused K:-1 red cell populations became strongly K:1 during a terminal episode of sepsis due to a group D streptococcus organism, Streptococcus faecium. Subsequent in vitro studies using normal K:-1 red cells inoculated with that organism showed that it could render the red cells agglutinable by reagents containing IgG anti-K1. In addition, disrupted S. faecium organisms rendered Jkb-negative red cells agglutinable by those reagents.

Detailed methodology and implementation of a semiautomated serial dilution microtechnique for antimicrobial susceptibility testing.

The detailed methodology and implementation of a semiautomatic microtechnique for performing serial dilution antimicrobial susceptibility studies are described. Quantitative susceptibility studies to a battery of antimicrobials are performed routinely on all significant clinical isolates. Results are reported as the minimal inhibitory concentration in micrograms per milliliter of broth. Guidelines relating standard doses of antimicrobials with expected blood and urine levels are presented to facilitate the use of the quantitative data. This microtechnique is used to measure serum and other body fluid levels of antimicrobial agents to document the level attained with a specific course of therapy. This technique is highly reproducible and has a high correlation with, and is at least 10 times faster than, standard glass tube techniques.

Analysis of cost and accuracy of alternative strategies for Enterobacteriaceae identification.

Analysis of the cost of time and material required for the diagnosis of Enterobacteriacea isolates indicated that a conventional 17-tube (20-test) setup costs $7.98 per isolated identified. Using the API 20E, a similar identification cost $3.02. A conventional 7-tube (10-test) setup cost $3.60, whereas the comparable cost by 30% while increasing the number of isolate identified correctly by 3%. Other strategres using the API 20E or a deoxyribounclease test were also evaluated for cost and accuracy.

Development of a lysis-filtration blood culture technique.

A lysed-blood culture system that quickly lyses patients' blood near neutrality and is relatively noninjurious to more delicate pathogens such as Haemophilus influenzae and Bacteroides fragilis is reported. The lysing solution includes culture medium, 0.004 M sodium carbonate and bicarbonate, 0.04% Triton X-100,and 0.6% Rhozyme (a mixture of proteases). Most of the pathogens tested multiplied in the lysing solution. The lysed blood normally is immediately filtered. The membrane is transferred to culture broth. The greatest advantage realized from this blood culture technique is separation of pathogens from antibiotics, bactericidal antibodies, complement, opsonins, and phagocytic systems. Another advantage is the concentration of organisms into a small volume of clear medium for faster growth and visualization of growth. It was observed that both gram-negative and -positive organisms were attracted during filtration to the filter material and were not removed from it by backwashing with buffer. Thus, filter membranes with porosities much larger than would nominally be expected to retain bacteria retained all or part of light and heavy Escherichia coli and Staphylococcus aureus suspensions. Advantage may be taken of this phenomenon to use filters with larger pore sizes and avoid filter clogging by poorly lysed specimens. Porr lysis may result from addition of too much blood to the lysing solution, blood with elevated numbers of erythrocytes or leukocytes, or blood from some people whose blood is naturally more resistant to lysis.

Phage pattern-specific oxacillin-resistant and borderline oxacillin-resistant Staphylococcus aureus in U.S. hospitals: epidemiological significance.

For a 13-year period (1978 through 1990), oxacillin-resistant (MIC, greater than 4 micrograms/ml) Staphylococcus aureus (ORSA) strains were collected from Clinical Center (National Institutes of Health) patients and patients from five other U.S. hospitals. From Clinical Center patients, 251 of 253 isolates (99%) were bacteriophage typed as phage group III. Five other hospitals contributed 203 ORSA strains, of which 188 (93%) were group III. The group III ORSA strains predominantly included a characteristic core pattern of phages, 7/47/53/54/75/77. For the low-level (borderline) oxacillin-resistant strains (MIC, 2 to 4 micrograms/ml), amoxicillin-clavulanic acid combination (Augmentin) testing disclosed 62 hyper-beta-lactamase producers, of which 59 (95%) were of a separate, distinct S. aureus strain, with the phage pattern 92/94/96/292/D-11 (group V). Thus, ORSA and hyper-beta-lactamase producing S. aureus are distinct epidemic strains.

Modified toluidine blue O stain for Pneumocystis carinii: further evaluation of some technical factors.

A modified toluidine blue O (TBO) stain for Pneumocystis carinii cysts was evaluated with regard to the influence of (i) the age and extent of use of the sulfation reagent, (ii) the source of TBO, (iii) the TBO content of the staining solution, and (iv) the amount of TBO present in the alcohol wash solutions. All TBOs evaluated, except for a new TBO obtained from Roboz Surgical Instrument Co., Inc., Washington, D.C., produced satisfactory results. Each lot of TBO should be quality controlled before use to ensure that the P. carinii cysts stain lavender against a blue background. We have ourselves decided to use only certified TBO with a high dye content. As extensively used sulfation reagent provided less satisfactory results than did either freshly prepared or 1-week-old unused sulfation reagent, we have decided to prepare fresh sulfation reagent at least weekly and to discard used sulfation reagent after 10 slides have been processed.

Lysis-filtration blood culture versus conventional blood culture in a bacteremic rabbit model.

Thirteen representative pathogenic bacterial species were used to create septicemia in rabbits, by injecting 10(6) colony-forming units into the marginal ear vein. At a selected time, usually 30 to 60 min after injection, heart blood was drawn into heparin and dispensed in 5.0-,0.5-, and 0.1-ml volumes into duplicate bottles of commercial brain heart infusion broth with sodium polyanetholesulfonate, and into duplicate bottles of a newly developed blood-lysing solution. Lysed blood was filtered, and the filter membranes were cultured in brain heart infusion broth. At the 5.0-ml blood inoculum level, of 126 total culture bottles (63 rabbits) for each system, 83 conventional cultures versus 109 lysis-filtration cultures were positive. At the 0.5-ml blood inoculum, 20 of 126 conventional culture bottles were positive, versus 66 of 126 lysis-filtration cultures. At the 0.1-ml blood inoculum, 2 of 126 conventional culture bottles were positive, versus 30 of 126 lysis-filtration cultures. Overall, 105 of 378 conventional cultures and 205 of 378 lysis-filtration cultures were positive. The advantage of the lysis-filtration system was striking for both gram-positive and gram-negative organisms at all inoculum concentrations, but was greater for gram-positive organisms. Most significant was the rate of recovery by this new system, when the number of bacteria in the blood was reduced to the point where recovery by conventional culture was unlikely. It is postulated that the superiority of lysis-filtration culture may be due to release of bacteria by lysis of phagocytes, preventing continued loss of pathogens by intracellular destruction during the first hours of blood culture.