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.

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.

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.

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.

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.

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.

Advantages of a modified toluidine blue O stain and bronchoalveolar lavage for the diagnosis of Pneumocystis carinii pneumonia.

A modified toluidine blue O staining technique for Pneumocystis carinii is described. An easily prepared sulfation reagent made with sulfuric and acetic acids was used. The stain can be employed for bronchoalveolar lavages and lung tissue touch preparations. Most background material was removed by the sulfation reagent, slides were generally easy to read, and time from receipt of a specimen to reporting of results was approximately 1 h. P. carinii cysts were more easily visualized with this stain than in slides stained with modified methylene blue, Gram, Gram-Weigert, and standard toluidine blue O procedures. The importance of certain procedural aspects of subsegmental bronchoalveolar lavage, by which most of the specimens were obtained, is also emphasized.

Evaluation of three bacteriuria screening methods in a clinical research hospital.

In a study conducted to compare three screening methods for their ability to detect significant bacteriuria, 2,815 urine specimens were screened by Chemstrip LN (BioDynamics, Division of Boehringer Mannheim Chemicals, Indianapolis, Ind.), 1,000 were screened by Bac-T-Screen (Marion Scientific Laboratory, Kansas City, Mo.), and 289 were screened by ATP assay (Turner Designs, Mountain View, Calif.). Results were compared with those obtained by quantitative culture plate method. The ATP assay showed the highest sensitivity (91%) compared with the Bac-T-Screen (67%) and Chemstrip LN (50%) tests but had the lowest specificity (64%) compared with the Bac-T-Screen (83%) and Chemstrip LN (91%). In 101 leukopenic patients with significant bacteriuria, the Bac-T-Screen test showed a higher sensitivity (33% at 10(4) to 10(5) CFU/ml and 80% at greater than or equal to 10(5) CFU/ml). It is concluded from this study that none of the three methods are sufficiently sensitive for the clinical research patients in this institution.

Comparison of lysis-centrifugation with lysis-filtration and a conventional unvented bottle for blood cultures.

Evaluation of a commercially available lysis-centrifugation blood culture system (Isolator, DuPont Co., Wilmington, Del.) and a lysis-filtration blood culture system for 3,111 cultures showed that both methods had comparable recoveries (73 and 68%, respectively) of significant aerobic and facultatively anaerobic isolates. The unvented conventional blood culture bottle had a recovery rate of 59%. Although the lysis-centrifugation and lysis-filtration systems had comparable recoveries of pathogens, the lysis-centrifugation system had the advantage of having colonies immediately available for further testing. The contamination rate with the lysis-centrifugation system was 3%, compared with 6% with the lysis-filtration system and 0.4% with brain heart infusion.

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.

Evaluation of four gentamicin and tobramycin assay procedures for clinical laboratories.

Accuracy, precision, and clinical laboratory utility of the TDX (Abbott Laboratories), Auto-ICS (Beckman Instruments, Inc.), COBAS-Bio (Roche Analytical Instruments, Inc.) with reagent kits (Syva), and EMIT (Syva) for gentamicin and tobramycin serum assay were assessed. TDX, COBAS-Bio, and EMIT analytical systems showed a proportional bias of less than 10% for recovery studies and a coefficient of variation less than 5% for within-run precision. The results of the recovery studies with the Auto-ICS showed a proportional bias of 25% with gentamicin and 16% with tobramycin. The within-run precision expressed as the coefficient of variation for the Auto-ICS was 6.7% for gentamicin and 8.6% for tobramycin. In comparisons involving gentamicin- and tobramycin-containing patient samples, the results with the TDX analytical system showed the best agreement with the COBAS-Bio. For the determination of these two antibiotics, the TDX analytical system provided the best overall accuracy and precision.

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.

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.

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.

Evaluation of five gentamicin assay procedures for clinical microbiology laboratories.

Five gentamicin assay procedures (a bioassay, an enzyme immunoassay, a latex agglutination inhibition test, a fluorescence immunoassay, and a radioimmunoassay) were evaluated to determine which was optimal for our laboratory. The evaluation was based on recovery and precision studies and results of analyses of patient samples, as well as technical assay performance factors. The latex agglutination inhibition test appears useful for laboratories performing only occasional assays for gentamicin; however, the fact that some rheumatoid factor-positive sera, as well as some other sera for unknown reasons, may give falsely low values is a potential drawback to this procedure. Because of its accuracy, precision, rapid turn-around time, and relative simplicity of performance, we selected the enzyme immunoassay procedure for routine use for gentamicin assays in our laboratory.

Evaluation of serum gentamicin assay procedures for a clinical microbiology laboratory.

Four methods for the measurement of serum gentamicin concentration were evaluated with respect to cost-effectiveness, accuracy, and precision. Gentamicin concentration was determined in 112 clinical samples by the Staphlococcus epidermidis agar diffusion bioassay procedure in routine service in our laboratory at the time this study was initiated. Appropriate portions of these clinical samples were frozen and later thawed for remeasurement of gentamicin by bioassay or for measurement of gentamicin in one of three other systems. These included the Enzymatic Radiochemical Assay, the Diagnostic Products Corporation Radioimmunoassay and the New England Nuclear Corporation Radioimmunoassay. In addition, gentamicin dissolved in horse serum at 2, 4, 6, 8, 10, and 12 micrograms/ml was aliquoted, frozen, and later thawed for assay in each of the above systems. The data were analyzed for evidence of constant and proportional bias as well as for accuracy and precision.

Computer analysis of Staphylococcus aureus phage typing data from 1957 to 1975, citing epidemiological trends and natural evolution within the phage typing system.

Computer analysis of Staphylococcus aureus phage ty ping data collected for over 18 years in a large research hospital showed a drastic decrease in the number of hospital epidemic strains. Phage lysis patterns gradually modified from those of earlier years and were a reflection of changes within the S. aureus reservoir, and not within the typing phages, since the typing phages were used from stable lyophilized stocks. There was increasing cross-lysis of S. aureus strains by phages of lytic groups I, II, and III, such that this grouping was no longer epidemiologically valid. A 61% increase in unique strains occurred from the period 1957 to 1975. Disappearance of the widely recognized epidemic strains was followed by a proliferation of unique strains with individual phage patterns. These increased from 38% in the period 1957 to 1962 to 62% in the period 1969 to 1975, indicating a trend toward a "one patient-one strain" situation. Nontypable strains decreased in more recent years from 16% (1957 to 1975) to 7% in 1978, following introduction of phages 94, 96, 292, and D-11. Pandemic S. aureus strain 80/81 first appeared in this hospital in 1959, 5 years after it was first reported in the United States. Strain 80/81 disappeared from the hospital in 1963, partly due to the advent of methicillin.

Microbiology subsystem of a total, dedicated laboratory computer system.

The computer system used by the Microbiology Service of the Clinical Pathology Department, Clinical Center, National Institutes of Health is discussed. This microbiology subsystem is a part of a dedicated on-line laboratory computer system used by the entire department. The laboratory computer is connected on-line to a hospital computer which provides patient admission, transfer, and discharge data. Mark sense worksheets and cathode ray tube terminals are used for result entry and correction. Cumulative patient reports are printed. Results for both active and completed accessions can be easily retrieved on cathode ray terminals in the laboratory. All laboratory data are archived on magnetic tape from which a research data base and microfiched laboratory records are generated. The manner in which the system is integrated in the routine operation of the microbiology laboratory is emphasized. In addition, some of the costs, benefits, liabilities, and pitfalls associated with the introduction of the computer in the laboratory are reviewed. Finally, we have presented our concept of some of the future enhancements to our present system and some of the directions in which any future microbiology system might develop.