Multicenter international work flow study of an automated polymerase chain reaction instrument.
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Autoanalysis↗
Biomedical subjects
D L Jungkind
Publications and source records attributed to D L Jungkind.
Vancomycin-dependent Enterococci: a clinical and laboratory assessment.
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Anti-Bacterial Agents↗
Evaluation of Amplicor PCR for direct detection of Mycobacterium tuberculosis from sputum specimens.
We evaluated the Amplicor PCR assay (Roche Molecular Systems, Branchburg, N.J.) for direct detection of Mycobacterium tuberculosis in sputum. A total of 532 specimens from 270 patients were decontaminated and stored at 4 or -75 degrees C until assayed by PCR. This assay used three-step sample preparation, biotinylated primer pairs, AmpErase, and a microtiter format for amplicon capture and detection. Amplicor PCR results were compared with clinical history, culture from a Lowenstein-Jensen slant, and results from the BACTEC TB-460 system. Eighty-seven cultures from 15 patients grew M. tuberculosis; of these, 83 (95%) were positive with the Amplicor PCR test. The false negatives were most likely due to sample variation and inhibitors. Of the 445 specimens from which M. tuberculosis was not isolated, 428 (96%) were negative with the Amplicor PCR test. Of the 17 M. tuberculosis culture-negative, Amplicor-positive specimens, 15 were reclassified as true positives because previous cultures grew M. tuberculosis. Of the 445 specimens which did not grow M. tuberculosis, Mycobacterium spp. other than M. tuberculosis were isolated from 150 specimens. Three of these 150 specimens were Amplicor positive; two were from a patient with a history of tuberculosis, and one specimen gave a false-positive result. We do not feel that this represents cross-reactivity, because repeated Amplicor testing of the isolate gave negative results. The microtiter plate has 96 wells. Allowing for six controls, 90 decontaminated specimens can be tested by one technologist in 7.5 h. This PCR assay took 7.5 h to complete and is a sensitive and specific, rapid method for the direct detection of M. tuberculosis from sputum.
Case Management↗
Urinary tract infection with an Enterococcus faecalis isolate that requires vancomycin for growth.
OBJECTIVE: To characterize the nutritional requirements and potential origin of a fastidious urinary tract Enterococcus faecalis isolate that apparently requires the antimicrobial agent vancomycin to grow. DESIGN: Case report and detailed microbiologic and molecular epidemiologic analysis. SETTING: University teaching hospital. MEASUREMENTS: Growth of the vancomycin-dependent strain was monitored using various standard laboratory media with and without supplementation with vancomycin and other substrates. This strain was compared with other vancomycin-resistant but nondependent E. faecalis strains by examining plasmid profiles and pulsed-field gel electrophoresis patterns of genomic DNA and by analyzing vancomycin-resistance genes identified by the polymerase chain reaction. RESULTS: An E. faecalis isolate, strain TJ310, was isolated repeatedly from the urine of a patient receiving long-term vancomycin therapy. This strain grew in primary culture but not on subculture, suggesting an unusual growth requirement, and ultimately was found to require the glycopeptide antibiotic vancomycin to grow. Strain TJ310 appeared to be closely related to other vancomycin-resistant but nondependent E. faecalis isolates with the vanB genotype previously isolated from the same patient, suggesting that vancomycin dependence may have evolved in vivo in a vancomycin-resistant enterococcal strain during continuous exposure to high concentrations of vancomycin in the urine. CONCLUSIONS: This is the first reported example of a clinical bacterial isolate that requires an antimicrobial agent to grow.
Drug Resistance, Microbial↗
A randomized, prospective trial comparing amoxicillin and erythromycin for the treatment of Chlamydia trachomatis in pregnancy.
OBJECTIVE: Our purpose was to evaluate the efficacy of amoxicillin as an alternative therapy to erythromycin for the treatment of cervical chlamydial infections during pregnancy. STUDY DESIGN: A randomized, prospective trial of two treatment regimens for Chlamydia trachomatis was performed in a cohort of pregnant women enrolled for care in an inner-city, university-based prenatal clinic, with an alternate-therapy crossover arm for primary treatment failures. Pregnant women diagnosed with chlamydial infection by McCoy cell culture of cervical swabs were assigned to receive either 500 mg of amoxicillin three times daily or 500 mg of erythromycin four times daily for 7 days. Patients' partners were treated with doxycycline. Compliance information was obtained by a standardized questionnaire at a posttherapy follow-up visit. Patients with positive follow-up cultures were crossed over into the alternate treatment arm and recultured at a later visit. RESULTS: During the study period 74 women consented to participate in this trial; 36 were treated with amoxicillin and 38 with erythromycin. Initial cure rates of 82.3% (28/34) for the amoxicillin group and 84.6% (27/32) for erythromycin were obtained before crossover (p = 0.91); four patients in each group were lost to follow-up. Overall cure rates after crossover were 84.6% (33/39) for amoxicillin and 84.2% (32/38) for erythromycin (p = 0.83). In the amoxicillin group 12.8% of patients reported side effects compared with 31.6% treated with erythromycin (p = 0.09), although seven erythromycin-treated patients compared with none of those in the amoxicillin arm stopped therapy because of side effects (p = 0.02). CONCLUSION: Amoxicillin offers a reasonable alternative to erythromycin for the treatment of Chlamydia trachomatis in pregnancy, on the basis of both cure rates and patient compliance.
Amoxicillin↗
Incidence of bacteremia associated with chorionic villus sampling.
OBJECTIVE: To assess the frequency of transient bacteremia among women undergoing transabdominal and transcervical chorionic villus sampling (CVS). METHODS: One hundred fourteen women undergoing CVS consented to participate in a university review board-approved study protocol. Exclusion criteria included known cardiac valve anomaly or replacement (or other prosthetic) and antibiotic use within the preceding 21 days. Blood cultures (aerobic and anaerobic) were drawn by a single operator on all patients, before CVS and within 15 minutes after completing CVS. Either the catheter tip or needle tip aspirate from each procedure was also sent for culture. RESULTS: Post-procedure bacteremia was detected in two (1.8%) of the patients undergoing CVS. These two patients both had their procedures performed transcervically, resulting in a 4.1% (two of 49) bacteremia rate after transcervical CVS, compared to none (zero of 65) in the transabdominal group (P = .36). The incidence of positive cultures from sampling instruments was also higher in the transcervical group (16.3 versus 0%; P = .003), but did not result in comparable rates of bacteremia among patients with positive instrument cultures. CONCLUSIONS: In this study, CVS was associated with a low rate of bacteremia, regardless of the procedure route. Recommendations for antibiotic prophylaxis in women with abnormal cardiac valves should parallel those for spontaneous vaginal delivery and other comparable genitourinary procedures.
Bacteremia↗
Systemic antimicrobial therapy for skin and skin structure infections: comparison of fleroxacin and ceftazidime.
Intravenous fleroxacin, 400 mg once a day, was compared with ceftazidime, 0.5-2 g three times a day or 1-2 g twice a day, for the treatment of skin and skin structure infections. Duration of treatment was 4-21 days. The study was a multicenter, unblinded comparison. Of the 316 patients enrolled, 212 were randomized to treatment with fleroxacin and 104 to ceftazidime (2:1 ratio); 92 fleroxacin-treated patients and 50 ceftazidime-treated patients were included in the standard analysis of efficacy. The most common diagnoses were wound infections and cellulitis, which affected 36% and 30% of the fleroxacin group, and 24% and 24% of the ceftazidime group, respectively. In the fleroxacin group, 82% of the infecting organisms were eradicated, and in the ceftazidime group, 79%. The overall rates of bacteriologic cure, by infection, were 79% for the fleroxacin group and 74% for the ceftazidime group, and those for clinical cure were 82% and 73%, respectively. It could not be concluded with 95% confidence that the two regimens resulted in equivalent cure rates because the range of between-group differences was outside the stipulated limits of +/- 15%. The percentage of patients with one or more adverse events was approximately twice as high (17% vs. 9%) in the fleroxacin group than in the ceftazidime group. The most frequent event in both groups was nausea. In this study, intravenous therapy with fleroxacin or ceftazidime produced similar bacteriologic and clinical cure rates, but the statistical requirements permitting a conclusion of equivalence at protocol levels were not met.
Adolescent↗
Clinical evaluation of a new polymerase chain reaction assay for detection of Chlamydia trachomatis in endocervical specimens.
A clinical evaluation of the Amplicor polymerase chain reaction (PCR) assay for the detection of Chlamydia trachomatis in endocervical swabs (Roche Molecular Systems, Branchburg, N.J.) is described. This new clinical system used one-step sample preparation, amplification with biotinylated cryptic plasmid primer pairs (CP24-CP27), uracil-N-glycosylase (AmpErase), and a microtiter format for amplicon capture and detection. Culture with McCoy cells in duplicate 1-dram (3.697-ml) vials with fluorescent immunostaining was the reference system. Endocervical swab samples from 945 women provided 74 culture-positive specimens, of which PCR detected 71. The initial PCR result was positive for 12 additional specimens. Arbitration of the PCR-positive, culture-negative samples by PCR with major outer membrane protein primers, duplicate culture, elementary body direct fluorescent-antibody staining, and DNA extraction PCR showed that all 12 samples were positive for chlamydia, raising the number of truly positive samples from 74 to 86. After arbitration the true sensitivities of PCR and culture were 96.5 and 86%, respectively (P = 0.02). Specificities for both were 100%. For PCR, the positive and negative predictive values were 100 and 99.7%, respectively. Total test efficiency was 99.7%. A high-test-volume (121 samples) timing study with all items included in the College of American Pathologists work load method indicated that this PCR format took approximately 3 min per sample. Because of the high sensitivity, specificity, and improved ease of handling, we found PCR to be a good alternative to culture for detection of C. trachomatis.
Cervix Uteri↗
Systemic antimicrobial therapy in skin and skin structure infections: comparison of temafloxacin and ciprofloxacin.
Temafloxacin is an investigational fluoroquinolone with in vitro activity against common skin pathogens and a favorable pharmacokinetic profile. A randomized, double-blind, multicenter study was conducted in 492 patients to compare the safety and efficacy of temafloxacin 600 mg with ciprofloxacin 750 mg for the treatment of bacterial infections of the skin or skin structure. Both drugs were given twice daily for 7-28 days. The most common diagnoses were abscess and superficial skin infection, which were usually caused by staphylococci. In evaluable patients, clinical success (cure plus improvement) rates were 96% in the temafloxacin group and 99% in the ciprofloxacin group. Bacteriologic eradication rates were 95% and 93% in the temafloxacin and ciprofloxacin groups, respectively. Both regimens were well tolerated. Temafloxacin appears to be safe and effective for the management of bacterial infections of the skin and skin structure.
Adolescent↗
Non-radioactive DNA probe for the rapid identification of Mycobacterium avium complex from clinical specimens.
We evaluated an alkaline phosphatase labelled oligonucleotide probe (SNAP(TM), Syngene Co., Molecular Biosystems, Inc., San Diego CA) for the direct culture identification of Mycobacterium avium complex (MAC) isolated from clinical specimens. Mycobacterial species identified by conventional biochemical methods were retested with this DNA probe using the Centri-Dot(TM) format. The probe accurately identified all 69 pigmented M. avium complex and 15 non-pigmented isolates of M. avium complex. There were no false-positives with 45 non-MAC mycobacteria isolates (10 species) and 16 non-mycobacteria organisms (10 species). The sensitivity and specificity of the SNAP(TM) culture identification for M. avium complex were 100%. The alkaline phosphatase labelled DNA probe is stable for at least 9 months. The procedure can be completed within 2 h and is easily adapted in the clinical laboratory. For the strains encountered in our laboratory, we conclude that the SNAP(TM) hybridization is a rapid, specific, and reliable method for culture identification of M. avium complex.
Alkaline Phosphatase↗
Failure of dithiothreitol and pronase to reveal a false-positive cryptococcal antigen determination in cerebrospinal fluid.
A patient with squamous cell carcinoma of the lung and a serum rheumatoid factor (RF) of 1:1,280 had a positive cerebrospinal fluid (CSF) latex agglutination test (LAT) for cryptococcal antigen, in culture-negative, India-ink-negative CSF. Pretreatment of the sample of CSF with 2-mercaptoethanol (2-ME) ablated the antigen titer and established the presence of a false-positive LAT, whereas CSF pretreated with dithiothreitol (DTT) and pronase continued to yield a false-positive result. The differing ability of pronase, DTT, and 2-ME to eliminate interfering substances from CSF has not been previously described. Moreover, because RF is unlikely to cross the blood-brain barrier, the authors postulated that malignant disease was responsible for the patient's false-positive LAT in CSF. Hence, the authors report the case to emphasize that false-positive LAT results in CSF are unlikely to be produced by RF and to underscore the benefit of using enzymatic and sulfhydryl-reducing agents when the validity of the initial test results are in doubt. Such a procedure will optimize the chances of accurately identifying false-positive LAT results in CSF.
Aged↗
Treatment of cutaneous infections. Worldwide experience with ciprofloxacin.
Oral ciprofloxacin, a new fluorinated 4-quinolone, was evaluated in 795 patients with skin and skin structure infections. Ninety-four point one percent of the patients treated had clinical success with eradication of almost all of the offending bacteria. Ciprofloxacin has good activity against gram-positive organisms and excellent activity against gram-negative organisms. Side effects were few and not considered significant.
Adolescent↗
Attempted reduction of Abbott MS-2 false-susceptible errors for cefotaxime-resistant Pseudomonas aeruginosa by medium modification.
Thirty-five modifications of Abbott MS-2 growth medium were evaluated in an attempt to reduce the high frequency of false-susceptible errors produced by the MS-2 system when testing resistant Pseudomonas aeruginosa against cefotaxime. A hypotonic modification with 0.1% KNO3 reduced the errors from 41 to 20% for 23 resistant isolates. All hypertonic modifications proved unsatisfactory. No modification reduced false-susceptible errors to acceptable clinical levels.
Cefotaxime↗
Simultaneous production of rhamnolipids, 2-alkyl-4-hydroxyquinolines, and phenazines by clinical isolates of Pseudomonas aeruginosa.
Of 72 clinical isolates of Pseudomonas aeruginosa examined for simultaneous production of secondary metabolites, 86% produced 2-alkyl-4-hydroxyquinolines, 75% produced rhamnolipids, and 58% produced phenazines, including pyocyanin. Whereas isolates producing two or one constituted smaller groups, 39% released all three metabolites. Metabolite production did not appear to influence site of infection.
Glycolipids↗
Intestinal fluke infection as a result of eating sushi.
Severe diarrhea in a female outpatient was caused by an intestinal fluke, identified as Heterophyes heterophyes, a natural parasite of humans and domesticated and wild fish-eating mammals. This parasite is endemic in the Orient and the Middle East. A detailed case history revealed that the woman had never traveled outside the continental United States but became infected while eating raw fresh-water fish (sushi) that had been served at a local Japanese restaurant. The restaurant specialized in serving a great variety of fresh-water and salt-water fish that were flown in from the Orient and other parts of the world. The authors' findings indicate that a person does not have to travel to an endemic area to become infected with this organism.
Diarrhea↗
Effect of using heat-inactivated serum with the Abbott human T-cell lymphotropic virus type III antibody test.
The Abbott enzyme immunoassay (Abbott Laboratories, North Chicago, Ill.) for human T-cell lymphotropic virus type III (HTLV-III) antibody was evaluated to determine the effect of using heat-inactivated (56 degrees C for 30 min) serum as the sample. Each of 58 nonreactive serum samples gave a higher A492 value when tested after heat inactivation. Ten of the samples became reactive after heating. Heat-inactivated serum should not be used in the current Abbott HTLV-III antibody test, because this can cause false-positive results.
Acquired Immunodeficiency Syndrome↗
Comparison of two commercially available test methods with conventional coagulase tests for identification of Staphylococcus aureus.
The API STAPHase (Analytab Products, Inc., Plainview, N.Y.) and SeroSTAT Staph (Scott Laboratories, Fiskville, R.I.) tests were compared to the conventional tube coagulase test and a slide coagulase test by using fresh isolates of members of the family Micrococcaceae. The 4-h, 24-h, and combined readings of the tube coagulase test detected 94.5, 99.5 and 100%, respectively, of 219 Staphylococcus aureus isolates. The API STAPHase, SeroSTAT Staph, and slide coagulase tests detected 95.9, 95.4 and 95.9% of the isolates of S. aureus, respectively. There were no false-positive results with any of the systems when tested with 103 strains of members of the family Micrococcaceae other than S. aureus. We concluded that the STAPHase and SeroSTAT Staph tests were equal in accuracy to the slide coagulase and 4-h tube coagulase tests and were suitable for use in the clinical microbiology laboratory. However, SeroSTAT Staph gave faster results than the API STAPHase, and the test was easier to perform. Also, the false-negative rate was high enough with the STAPHase, SeroSTAT Staph, and the slide coagulase tests that all negative reactions should be confirmed with a tube test.
Bacteriological Techniques↗
False-susceptible results from the MS-2 system used for testing resistant Pseudomonas aeruginosa against two third-generation cephalosporins, moxalactam and cefotaxime.
The MS-2 system (Abbott Laboratories) was used for cefotaxime and moxalactam susceptibility testing of 111 Pseudomonas aeruginosa isolates collected from our burn treatment center. The frequency of very major errors was 52% for moxalactam and 32% for cefotaxime for all isolates requiring a minimal inhibitory concentration of greater than or equal to 64 micrograms/ml. For isolates requiring a minimal inhibitory concentration of 64 micrograms/ml, the very major errors were 78% for moxalactam and 57% for cefotaxime. Testing of an additional 44 isolates collected from another hospital confirmed that these results were not unique to nosocomial strains from within the burn center. The ratio of errors did not change when MS-2 results were compared with disk diffusion results. Colony count testing done on cuvettes which produced false-susceptible results indicated that little numerical change occurred during the 5- to 6-h test cycle, whereas microscopy of cuvette contents revealed metabolically active filamentous bacilli. The high frequency of false-susceptible results indicated that the MS-2 should not be used for testing clinical P. aeruginosa isolates for resistance to the two third-generation cephalosporins currently available for the MS-2. A comparison of the disk diffusion results indicated that neither moxalactam nor cefotaxime could be reliably used for predictive, class-concept testing of P. aeruginosa.
Cefotaxime↗