Detection of Mycobacterium tuberculosis in respiratory specimens by strand displacement amplification of DNA.

A total of 294 clinical respiratory specimens, including 75 with culture-positive results, were tested for the presence of Mycobacterium tuberculosis by strand displacement amplification (SDA) of DNA. A region of the IS6110 insertion element and an internal control sequence were amplified and then detected by a chemiluminescence assay. Receiver operator-characteristic curves were used to evaluate three methods for declaring specimens positive for M. tuberculosis. By the preferred method, SDA chemiluminescence results were converted to theoretical numbers of M. tuberculosis organisms. A positive threshold (PT) value, above which 95% of the SDA results were judged to be M. tuberculosis positive (sensitivity = 95%), was found to be 2.4 M. tuberculosis organisms per SDA reaction. The analogous PT value for 95% sensitivity on smear-positive specimens was 3.6 M. tuberculosis organisms per reaction. The PT of 2.4 M. tuberculosis organisms per reaction detected 100% of culture-positive, smear-positive specimens (sensitivity = 100%), while 95% sensitivity was achieved with a PT of 15.5 M. tuberculosis organisms per reaction. Specificities, which were calculated with respect to culture- and smear-negative specimens, ranged from 96% at a PT of 15.5 M. tuberculosis organisms to 84% at a PT of 2.4 M. tuberculosis organisms per reaction. The M. tuberculosis-negative specimens were also segregated according to whether the patients received antituberculosis chemotherapy. SDA specificity ranged from 90% (PT = 2.4 M. tuberculosis organisms) to 98% (PT = 15.5 M. tuberculosis organisms) for the M. tuberculosis-negative specimens from patients who had not received chemotherapy. SDA specificity in the M. tuberculosis-negative specimens from patients who received chemotherapy was lower (85 to 94%). This study represents the first large-scale demonstration of M. tuberculosis detection in clinical sputum specimens by isothermal DNA amplification with SDA.

Rendering of mycobacteria safe for molecular diagnostic studies and development of a lysis method for strand displacement amplification and PCR.

Two criteria must be met before mycobacterial specimens can be tested by DNA amplification methods: (i) the sample must be rendered noninfectious, and (ii) the organisms must be lysed to free the DNA. Previous publications reporting DNA amplification of mycobacteria have concentrated on lysis and amplification procedures and have not addressed the issue of sample safety. We have shown that heating of samples below 100 degrees C may not consistently kill mycobacteria; however, heating at 100 degrees C in a boiling-water bath or a forced-air oven for a minimum of 5 min kills mycobacteria, including Mycobacterium thermoresistibile. Furthermore, heating at 100 degrees C for 30 min consistently lyses mycobacteria to produce short fragments of DNA that are suitable for amplification by PCR and strand displacement amplification. This procedure works with clinical samples digested by the n-acetyl cysteine-NaOH method as well as with suspensions of organisms in phosphate buffer. This paper also demonstrates the feasibility of using strand displacement amplification with clinical specimens.

Comparison of Difco ESP and Organon Teknika BacT/Alert continuous-monitoring blood culture systems.

The Difco ESP and Organon Teknika BacT/Alert (BTA) systems were evaluated in a clinical study of 5,421 aerobic and 5,035 anaerobic blood cultures. Of 405 clinically significant positive cultures evaluated, 272 grew in both systems, 86 grew in ESP only, and 47 grew in BTA only (P < 0.005). Of 320 organisms detected in aerobic bottles, 208 grew in both systems, 68 grew in ESP only and 45 grew in BTA only (P < 0.05), with Staphylococcus aureus the only organism showing a statistically significant difference. The ESP anaerobic bottle also detected more anaerobes (16 of 17 versus 4 of 17, P < 0.005) and more organisms overall (57 versus 34, P < 0.05). However, with the exception of patients with anaerobic bacteremia (12 of 13 for ESP and 4 of 13 for BTA, P < 0.05), there was no statistical difference in the detection of patient episodes. Average detection time of matched aerobic bottles was 18.3 h for ESP and 22.0 h for BTA (P < 0.001). For matched pairs of anaerobic bottles, the average detection time was faster in the BTA bottles (P < 0.001), because of the growth of facultative organisms. To explore the differences in anaerobic detection more fully, 20 sets of anaerobic bottles were seeded with 12 anaerobic species mixed with human blood. ESP grew more organisms (17 of 20 versus 10 of 20, P < 0.025), and the average time to detection for the 10 paired positive cultures was 21.6 h for ESP and 50.8 h for BTA (P < 0.05). Times for loading and unloading bottles were similar for both systems.

Non-isotopic hybridization assays for bacterial DNA samples.

The background problem associated with the use of streptavidin in detecting biotin-labelled probes hybridized to DNA in crude bacterial extracts has been investigated. We have found that streptavidin binds specifically to a limited number of polypeptides which are difficult to remove by rapid extraction processes. Altering the hybridization and detection protocols results in a marked but not complete reduction of non-specific background in streptavidin-biotin assays. Complete elimination of non-specific background was achieved only when streptavidin was replaced with antibodies for the detection of biotinylated or sulphone-modified probes. The antibody-sulphone and streptavidin-biotin dot blot assays described here require 4.5-5 hours to perform and can detect DNA sequences in samples extracted from 2 x 10(7) cells or fewer.

Pseudomonas putrefaciens bacteremia.

Pseudomonas putrefaciens is an unusual cause of human disease. Since 1978 only five cases of bacteremia due to this organism have been reported. Within 12 recent months four cases of bacteremia due to P. putrefaciens were seen - two occurred in patients with chronic infections of a lower extremity, one in a patient with neutropenia, and one in a patient with fulminant septicemia and disseminated intravascular coagulation. Two of the patients had prostheses; in neither case did prosthetic infection or prosthetic failure occur. Two syndromes of bacteremic infection with P. putrefaciens are suggested. One syndrome is associated with chronic infection of a lower extremity, is fairly well tolerated, and responds to appropriate antimicrobial agents. The other syndrome is more fulminant and may be associated with severe underlying debility, liver disease, and malignancy. It is not yet known whether this is a meaningful distinction. The significance of the recent increase is the isolation of this organism is not clear at present.

Multicenter comparison of MicroScan and BACTEC blood culture systems.

Recently, MicroScan (Baxter MicroScan Div., W. Sacramento, Calif.) introduced a radiolabeled-blood-culture system that is compatible with the BACTEC 460 (Johnston Laboratories, Inc., Towson, Md.). A multicenter blood culture study was initiated to evaluate this new system. Approximately 20 ml of blood was obtained from each patient and divided equally between BACTEC and MicroScan bottles which were incubated and processed identically. Aerobic bottles were examined twice on days 1 and 2 and once on days 3, 4, 5, 6, and 7. Anaerobic bottles were examined once a day for 7 days. There were 3,451 cultures evaluated, and 414 of these subsequently grew microorganisms. Of these positive cultures, 64 were judged to be contaminated. Of the remaining 350 positive cultures, 253 grew in both systems, 54 grew in BACTEC bottles only, and 43 grew in MicroScan bottles only. The average times to detect positive cultures were 1.8 and 2.1 days by the BACTEC and the MicroScan systems, respectively. No significant difference in the number or kind of organisms recovered or in the detection times for positive cultures was observed between the two blood-culturing systems.

Nucleic acid probes in clinical microbiology.

The infectious disease applications of nucleic acid probe have been described. In addition, the basic procedures of nucleic acid probe technology have been discussed, as have the factors affecting implementation of probe technology in diagnostic laboratories. Despite the questions raised, nucleic acid probes will become part of the diagnostic laboratory in the near future. Commercial interests are developing and marketing new probes, reagents, and kits which will expedite the employment of this technology. High-volume reference laboratories will first use probes as part of a battery of tests which will include ELISA and monoclonal antibody methods. In all probability, probes will replace methods: that have proven to be ineffective, difficult, or costly such as culturing for some enteric pathogens and Legionella, that require long incubation periods, such as mycobacteria, or that have high costs and low yields, such as virology.

Pseudomonas alcaligenes endocarditis.

Pseudomonas alcaligenes is a common soil and water inhabitant that has rarely been proven a human pathogen. We describe a fatal case of Pseudomonas alcaligenes endocarditis. The need for accurate identification of unusual organisms isolated in a clinical setting are discussed.

Bacterial prostatitis caused by Staphylococcus saprophyticus.

Staphylococcus saprophyticus has been shown to be an important uropathogen in urinary tract infections in young women. Thirty-five isolates from 27 patients with staphylococci in the prostatic fluid of men with bacterial prostatitis were evaluated for the presence of S. saprophyticus. Three patients (11 per cent) with this organism were identified by novobiocin resistance (disk diffusion test), absence of hemolysis, and coagulase. These patients tended to be younger, more symptomatic, and more responsive to appropriate antibiotic therapy than those with staphylococcus epidermidis. S. saprophyticus appears to be an important pathogen in prostatic infections.

The value of the sputum gram's stain in community-acquired pneumonia.

Expectorated sputum Gram's stain was correlated with clinical presenting data, cultures, serological data, and response to antibiotic therapy in 89 patients admitted with community-acquired pneumonia. The finding of Gram-positive diplococci on Gram's stain correlated with brief antecedent illness, Streptococcus pneumoniae growing from cultures, and a rapid response to single-agent antibiotic therapy. Patients with no predominant pathogen on Gram's stain were clinically indistinguishable from those with Gram-positive diplococci except for a longer antecedent illness, lack of growth of pathogens from cultures, and more prolonged response to antibiotic therapy. Sputum cytological findings and transtracheal cultures were useful in patients with no Gram-positive diplococci on Gram's stain. The sputum Gram's stain is proposed as a sensitive and reliable indicator to guide therapy and predict outcome in adults with community-acquired pneumonia.

Evaluation of the API 20C for identification of yeasts.

The API 20C reactions were compared with the results of conventional methods for identifying yeasts using 45 strains. There was a 98% (44/45) correlation between the two methods in the identification of the yeasts. Individual fermentation tests ranged from 89 to 100% correlation, with an average agreement of 96.7%. The assimilation tests ranged from 85 to 100% correlation, with an overall agreement of 97.3%. The two methods had 91% agreement for cycloheximide resistance. The results of this limited study indicate that the API 20C may prove to be an acceptable substitute for conventional carbohydrate fermentation and assimilation tests.

Haloprogin: a topical antifungal agent.

Haloprogin was shown to be a highly effective agent for the treatment of experimentally induced topical mycotic infections in guinea pigs. Its in vitro spectrum of activity also includes yeasts, yeastlike fungi (Candida species), and certain gram-positive bacteria. The in vitro and in vivo antifungal activity of haloprogin against dermatophytes was equal to that observed with tolnaftate. The striking differences between the two agents were the marked antimonilial and selective antibacterial activities shown by haloprogin, contrasted with the negligible activities found with tolnaftate. Addition of serum decreased the in vitro antifungal activity of haloprogin to a greater extent than that of tolnaftate; however, diminished antifungal activity was not observed when haloprogin was applied topically to experimental dermatophytic infections. Based on its broad spectrum of antimicrobial activity, haloprogin may prove to be a superior topical agent in the treatment of dermatophytic and monilial infections in man.