Detection of Bordetella pertussis by rapid-cycle PCR and colorimetric microwell hybridization.

The use of rapid-cycle PCR combined with colorimetric microwell hybridization for detecting Bordetella pertussis was investigated. Rapid cycling was performed with an air thermocycler (model 1605; Idaho Technology, Idaho Falls, Idaho). Although the instrument was originally designed to be used with capillary tubes, an adapter that allows this instrument to be used with PCR tubes has recently been introduced. Because of the low heat capacity of air, the thermocycler has rapid transition rates between temperatures. The combination of a rapid temperature transition rate, small sample volume (10 microliters), and overshooting or undershooting of the temperature set points allowed the cycles to be reduced to 5 s for denaturation and 10 s for extension and annealing. Thus, the amplification could be completed in a total of approximately 35 min. Amplified DNA was detected with biotin-labeled primers and by hybridization to a capture probe immobilized in microwell plates. When simulated clinical specimens consisting of pooled nasopharyngeal washes with known numbers of B. pertussis organisms were examined by this procedure, as little as one organism per 5 microliters of sample could be detected. Six nasopharyngeal aspirates or washes from culture-positive patients were positive by PCR, as were two of seven specimens obtained from patients that were negative by culture and direct fluorescent-antibody assay. The two patients who were PCR positive but culture and direct fluorescent-antibody assay negative had clinical disease compatible with pertussis. This method appears to be a sensitive, convenient means of detecting B. pertussis in clinical specimens. The total time required for specimen processing, amplification, and detection is about 2.5 h.

The polymerase chain reaction: a revolutionary new procedure for the laboratory diagnosis of infectious disease.

The polymerase chain reaction (PCR) is a revolutionary new means of amplifying, ie, replicating, selected DNA sequences in vitro. This procedure is rapid, requiring approximately 5 hours for completion, and exquisitely sensitive. Studies have shown that as few as one microorganism can be detected. Thus, it has the potential for revolutionizing the diagnosis of infections. Because of its expense, its immediate role will probably be restricted to infections where the causative organism cannot be cultured or is difficult to detect by conventional means. As further progress occurs, however, this technique may well become a major new tool for diagnosing infections.

Diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients by polymerase chain reaction (PCR).

Polymerase chain reaction (PCR) testing was performed on respiratory tract specimens obtained by throat swab in 21 children admitted to the hospital with suspected Mycoplasma pneumoniae pneumonia. Of 13 patients with a clinical condition compatible with mycoplasma infection and an immunological response to M. pneumoniae, 11 were positive by PCR. Eight patients were negative by serology and/or had a clinical condition not compatible with mycoplasma infection, and all were negative by PCR. The antibody response to M. pneumoniae was delayed for a week or more in 3 (23%) of the 13 patients with documented mycoplasma infection. These results suggest that PCR performed on a respiratory tract specimen obtained by a throat swab may be useful in the initial evaluation of children with suspected M. pneumoniae pneumonia, especially in patients in whom the serological response is delayed.

Rapid, sensitive detection of Mycoplasma pneumoniae in simulated clinical specimens by DNA amplification.

The polymerase chain reaction (PCR) was investigated as a means of diagnosing Mycoplasma pneumoniae infections. The target DNA sequence was a 375-bp segment of the P1 virulence protein. This DNA segment was amplified in pure cultures of five different strains of M. pneumoniae but not in other species of Mycoplasma, Acholeplasma, or Ureaplasma that were tested. Simulated clinical specimens were used to compare PCR, culture, and the gene probe. The sensitivity of PCR was between 1 and 10 organisms. The sensitivity of culture was approximately 10(3) organisms, and the gene probe detected between 10(4) and 10(5) organisms. These results indicate that PCR has significant potential as a rapid, sensitive method for detecting M. pneumoniae in clinical specimens.

Rapid, simple method for treating clinical specimens containing Mycobacterium tuberculosis to remove DNA for polymerase chain reaction.

Several simplified methods for treating mycobacteria to release DNA for amplification by the polymerase chain reaction (PCR) were investigated. The most effective of the methods was sonication. Samples were placed in screw-capped microcentrifuge tubes that were then placed in a plastic rack. The rack was floated in a dish of water next to the ultrasonic probe so that the ultrasonic energy was transmitted through the walls of the tubes. This allowed multiple samples to be processed safely and effectively. Forty-three clinical samples were processed by this procedure, and the crude preparations were analyzed for Mycobacterium tuberculosis by PCR. Twenty-six of these specimens contained M. tuberculosis, and 17 either had no growth or contained other species of mycobacteria. Twenty-four of the 26 (92%) positive specimens were correctly identified, and all of the negative specimens were correctly identified. This sonication procedure appears promising as a rapid, simple means of treating clinical specimens containing mycobacteria for PCR analysis.

Campylobacter pylori and gastroduodenal disease.

Campylobacter pylori is a newly described, spiral-shaped, gram-negative bacillus that is oxidase positive, catalase positive, and urease positive and grows slowly in culture. Although observed in human tissue at the beginning of the century, it was not cultured until 1982. Because there are significant morphological and genetic differences between this organism and other species of Campylobacter, it will probably be reclassified in a new genus. Current information indicates that the organism primarily resides in the stomach tissue of humans and nonhuman primates and may occasionally spread to the esophagus or other parts of the alimentary tract under appropriate conditions. Significant evidence has accumulated in the last several years to show that it causes gastritis, and there is mounting evidence that it may participate in the development of duodenal ulcers. It may also be associated with gastric ulcers and nonulcer dyspepsia. It can be detected in patients by culture of biopsy specimens or histological staining of biopsy tissue. Indirect evidence for the presence of the organism can be obtained by detection of urease in a tissue biopsy specimen, by urea breath tests, or by detection of specific antibody. It may not be necessary to implement these procedures for routine use, however, until the role of the organism can be defined better. Ultimately, the discovery of this organism may lead to radical changes in the diagnosis and treatment of gastric disease.

Nonculture methods for detection and identification of microorganisms in clinical specimens.

The pressures of cost reduction and clinical relevancy that led to the conception and introduction of direct detection tests continue to influence the course of modern microbiology. A significant amount of progress has been made in the past few years in the refinement of these tests. Some of the procedures that have been developed have been disappointing in their true clinical utility. Others seem to be a genuine advance in technique. Unquestionably, these methods will continue to be developed and will have a significant impact on the way laboratory testing is performed and, we hope, the cost of laboratory work and the quality of care given to patients.

Medium supplementation for growth of Campylobacter pyloridis.

Experiments were conducted to define the growth requirements of Campylobacter pyloridis, a newly discovered organism associated with gastritis and peptic ulcers. Two clinical isolates were streaked onto various media, and growth was assessed semiquantitatively according to relative colony size and extent of growth through the streak. The growth obtained on fresh chocolate agar, composed of GC agar base (Difco Laboratories, Detroit, Mich.) plus 1% hemin, was used as a reference. The organism grew on both GC agar base and Mueller-Hinton agar without supplementation, but less well than on chocolate agar. No growth occurred on tryptic soy or brucella agar. Supplementation of brucella agar with 1 or 5% horse serum or 0.1 or 1.0% cornstarch supported growth to about the same level as GC agar base alone. Supplementation with hog gastric mucin or methyl cellulose supported weak growth. GC agar base with 1% starch or 0.2% charcoal supported growth as well as chocolate agar. Experiments with brucella broth provided similar results. Cornstarch and methyl cellulose partially replaced the requirement for serum, but methyl cellulose and hog gastric mucin did not. These results show that some form of supplementation is necessary for growth of C. pyloridis. This can be starch, serum, charcoal, or hemin, but hemin is not an absolute requirement for growth.

Clinical comparison of the Roche Septi-Chek and Dupont Isolator blood culture systems.

A study was conducted to compare the recovery of clinical isolates by the DuPont Isolator and Roche Septi-Chek blood culture systems. A total of 5,262 blood culture specimens were processed by the two systems. Of these, 358 cultures contained significant isolates: 219 were positive in both systems, 68 were recovered only by Isolator, 71 were recovered by Septi-Chek only (not statistically significant). Of the isolates recovered in both systems, 159 were positive the same day, 55 were recovered first by Isolator, and 5 were recovered first by Septi-Chek. In cases where Isolator recovered organisms first, the average difference in time was one to two days. Regarding particular groups of organisms, there was no difference between the systems in recovery of Enterobacteriaceae, anaerobes, yeast, and gram-positive bacteria, except for Streptococcus pneumoniae. Septi-Chek recovered S. pneumoniae significantly more often. These results suggest that these two systems are essentially comparable, except with S. pneumoniae, although the Isolator frequently provided results more rapidly.

Performance of four slide agglutination methods for identification of Staphylococcus aureus when testing methicillin-resistant staphylococci.

Four commercially available slide agglutination systems for identifying Staphylococcus aureus were compared with the conventional slide (clumping factor) and tube coagulase tests. The systems evaluated included Bacto Staph Latex (Difco Laboratories, Detroit, Mich.), Staphyloslide (BBL Microbiology Systems, Cockeysville, Md.), Mini ID Accu-Staph (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.), and Staphaurex (Wellcome Diagnostics, Research Triangle Park, N.C.). A total of 338 clinical isolates, including methicillin-resistant S. aureus (n = 149), methicillin-susceptible S. aureus (n = 78), methicillin-resistant, coagulase-negative staphylococci (n = 45), and methicillin-susceptible, coagulase-negative staphylococci (n = 66), were tested by each method. The slide test for clumping factor, the 4-h tube coagulase test, Bacto Staph Latex, Staphyloslide, Mini ID Accu-Staph, and Staphaurex detected 212 (93.4%), 218 (96%), 223 (98.2%), 223 (98.2%), 221 (97.4%), and 224 (98.7%) of the S. aureus (44% methicillin-resistant) isolates, respectively. There were no false-positive results with any of the methods when the 111 strains of coagulase-negative staphylococci were tested. The results of this evaluation suggest that the four slide identification methods tested can provide rapid and accurate identification of methicillin-resistant S. aureus strains.

Relation of Campylobacter pyloridis to gastritis and peptic ulcer.

Biopsy specimens from the gastric antral mucosa of 50 patients with upper gastrointestinal complaints were studied by light and electron microscopy and culture. Of 46 assessable specimens, seven were histologically normal, and 39 showed evidence of gastritis. Twenty-seven of the specimens with evidence of gastritis (69%) contained spiral bacteria, whereas only one of the normal specimens (14%) contained these bacteria (P = .02). Of 17 patients found to have gastric ulcers, 10 (59% [P greater than .10] ) also had spiral bacteria. The bacteria could be seen scattered over the surface of the epithelial cells and just under the layer of mucus but were rarely found inside the epithelial cells. Curved or spiral gramnegative bacilli were isolated from 10 of the specimens on chocolate agar incubated at 37 C under microaerophilic conditions. The bacteria resembled the organism recently named Campylobacter pyloridis by other investigators.

Composition of the antigenic material removed from Campylobacter jejuni by heat.

The antigenic material removed form Campylobacter jejuni by the boiling of whole cells in saline was examined biochemically. Analyses showed that the extracted material contained 3 micrograms of protein per ml per mg of wet cells and ca. 2.6 micrograms of carbohydrate per ml per mg of wet cells. Further extraction of the material with chloroform-methanol produced about 0.5 microgram of water-insoluble residue per ml per mg of wet cells, suggesting the presence of lipid as well. Additional analyses revealed the presence of hexose, pentose, heptose, hexosamine, and 2-keto-3-deoxyoctonic acid, and the extract was also positive by the Limulus amoebocyte lysate assay for lipopolysaccharide. An examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that at least 10 different protein bands could be detected. One of the major bands corresponded to the major outer membrane protein, as determined by comparison with an outer membrane protein preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another major protein in the heated extract corresponded to a band previously shown to be flagellin. An analysis of the time course of the release of material showed that a significant amount was removed after 3 to 10 min at 100 degrees C, but the release of material seemed to be delayed at lower temperatures. These results show that the treatment of C. jejuni with heat produces a complex mixture of components, including cell wall lipopolysaccharide, the major outer membrane protein, and flagellin. It is likely that some cytoplasmic components are present as well. Blebs of outer membrane have been observed with this organism by electron microscopy. Our results confirm this and suggest that the heating of cells accelerates this blebbing process.

Clinical evaluation of a lysis-centrifugation technique for the detection of septicemia.

A commercially available lysis-centrifugation blood culture system was compared with a two-bottle broth-culture system employing 100 mL of broth and 10 mL of blood per bottle to analyze 1,913 blood specimens. Of 154 clinically significant isolates, 89% were detected by the lysis-centrifugation technique, and 73% were detected by the broth-culture method. Twenty-seven percent of the organisms were detected only by the lysis-centrifugation technique, and 11% were detected only by the broth system. Fifteen polymicrobial cultures were encountered; the lysis-centrifugation technique detected 93% of the organisms in these cultures, while the broth-culture method detected only 20%. Isolated colonies of clinically important organisms were available 30 hours earlier with the lysis-centrifugation technique. These results suggest that the lysis-centrifugation technique may provide a substantial improvement over conventional methods for blood cultures.

Evaluation of a lysis-centrifugation and biphasic bottle blood culture system during routine use.

An in-use evaluation of a commercially available lysis-centrifugation blood culture system (Isolator; Du Pont Co., Wilmington, Del.) is presented. The Isolator was compared with biphasic bottles containing Trypticase soy broth and agar for the detection of organisms in 3,129 paired blood samples. Of 272 potential pathogens recovered, 78% were detected by the Isolator system, and 69% were detected by the biphasic bottle. A total of 31% of these organisms were detected only by the Isolator, and 22% were detected only by the biphasic bottle. The Isolator demonstrated enhanced detection of facultative gram-negative bacilli, anaerobic bacteria, and polymicrobial cultures. The biphasic bottle was more effective for the recovery of facultative gram-positive cocci, especially Streptococcus pneumoniae. The two systems were equally effective for the recovery of yeasts. Contamination rates were 3% for the Isolator and 3.2% for the biphasic bottle. The results indicate that the Isolator system performs well in routine clinical use, but it should be complemented by another method to obtain optimal detection of bacteremia. The biphasic bottle provides an acceptable complementary system both in terms of utility and performance.

Electron microscopy of the coccoid form of Campylobacter jejuni.

Confluent cultures of Campylobacter jejuni incubated for 24 and 48 h each were examined by electron microscopy. Although the 24-h-old cells exhibited typical curved morphology, the 48-h-old cells showed rounded morphology with a loss of cell integrity. This appeared to be an autolytic process that occurred very rapidly after the culture became mature. These results confirm previous evidence that the coccoid form of this organism is a degenerate state.

Susceptibility testing of Campylobacter fetus subsp. jejuni, using broth microdilution panels.

Twenty-five isolates of Campylobacter fetus subsp. jejuni were tested by broth microdilution panels (Sensititre; GIBCO Diagnostics, Chagrin Falls, Ohio) and the minimal inhibitory concentrations (MICs) were compared with the corresponding MICs obtained by the standard agar dilution technique. Microdilution panels designed for testing gram-positive organisms were used so that erythromycin, the antibiotic of choice for this organism, could be included. The correlation with agar dilution was relatively poor when Mueller-Hinton broth was used; the MICs that were within one twofold dilution of the corresponding agar dilution MIC ranged from 15% with tetracycline to 75% with ampicillin. The overall agreement for all antibiotics tested was 48%. The correlation improved significantly, however, to an overall agreement of 87% when Wilkins-Chalgren broth was substituted in the broth microdilution procedure. Our results indicate that the broth microdilution test is an accurate method for testing this organism, provided than an appropriate medium is used.