The impact of the polymerase chain reaction in clinical medicine.
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Clinical Medicine↗
Biomedical subjects
G D Ehrlich
Publications and source records attributed to G D Ehrlich.
PCR-based detection of bacterial DNA after antimicrobial treatment is indicative of persistent, viable bacteria in the chinchilla model of otitis media.
PURPOSE: Bacterial deoxyribonucleic acid (DNA) has been previously detected by polymerase chain reactions (PCR) in a significant percentage of culturally-sterile pediatric middle-ear effusions. The current study was designed to determine whether this represents the existence of viable bacteria or the persistence of residual DNA in the middle-ear cleft. MATERIALS AND METHODS: The middle-ear cavities of two sets of chinchillas were inoculated with either: 1) 100 colony-forming units (CFU) of live Haemophilus influenzae, 2.2 x 10(6) CFU of pasteurized Moraxella catarrhalis, and 1000 ng of DNA (>10(8) genomic equivalents) from Streptococcus pneumoniae; or 2) 100 CFU of live S pneumoniae, 2.2 x 10(6) CFU of pasteurized M catarrhalis and 1000 ng of purified DNA from H influenzae. Animals were treated with ampicillin for 5 days beginning on day 3. A single-point longitudinal study design was used for sampling to eliminate the possibility of contamination. RESULTS: No DNA was detectable from the heat-killed bacteria or the purified DNA after day 3. However, DNA from the live bacteria persisted through day 21, even though all specimens were culture-negative following the initiation of antimicrobial therapy. CONCLUSION: These findings indicate that purified DNA and DNA from intact but nonviable bacteria do not persist in the middle-ear cleft in the presence of an effusion, even following high copy inoculation. In contrast, antibiotic-treated bacteria persist in some viable state for weeks as evidenced by the differential ability of the PCR-based assay systems to detect the live bacteria, but not detect the heat-killed organisms.
Ampicillin↗
Prevention of CMV disease in allogeneic BMT recipients by cytomegalovirus antigenemia-guided preemptive ganciclovir therapy.
PURPOSE: Cytomegalovirus (CMV) infection can cause severe disease and mortality in recipients of allogeneic bone marrow transplants (alloBMT) when either the donor or recipient is CMV seropositive (high-risk alloBMT). We investigated the efficacy of preemptive therapy guided by detection of CMV antigenemia. METHODS: In 11 high-risk alloBMT recipients, high-dose ganciclovir (GCV) treatment was initiated at first positive antigenemia and was continued until antigenemia became negative. RESULTS: The treatment strategy prevented CMV disease during the follow-up period of the study in 7 alloBMT recipients with positive CMV antigenemia. Three other patients who were shown to be CMV antigenemia negative but positive for CMV DNA in blood by the polymerase chain reaction (PCR) were not treated and did not develop CMV disease. The eleventh patient was negative for CMV by all tests for the duration of the study and did not develop CMV disease. CONCLUSIONS: We have found antigenemia-guided preemptive GCV therapy to be an effective strategy for the prevention of CMV disease in high-risk alloBMT recipients.
Adolescent↗
Molecular analysis of bacterial pathogens in otitis media with effusion.
OBJECTIVE: To determine if the polymerase chain reaction (PCR) can detect bacterial DNA in pediatric middle ear effusions that are sterile by standard cultural methods. DESIGN: Single-center, blinded, comparative study of diagnostic assays. The PCR-based detection systems for Moraxella catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae were designed and validated using a battery of DNAs obtained from cultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR. SETTING: Tertiary care pediatric hospital. PATIENTS: A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pittsburgh (Pa) during myringotomy and tube placement for chronic otitis media with effusion (duration > 3 months). All patients had failed multiple courses of antimicrobial therapy and were diagnosed by a combination of validated otoscopy and tympanograms. MAIN OUTCOME MEASURE: Differences in the percentage of positive test results between PCR-based assays and culture for M catarrhalis, H influenzae, and S pneumoniae. RESULTS: Of the 97 specimens of otitis media with effusion, 28 (28.9%) tested positive by both culture and PCR for M catarrhalis, H influenzae, or S pneumoniae. An additional 47 specimens (48%) were PCR positive/culture negative for these three bacterial species. Thus, 75 (77.3%) of the 97 specimens tested PCR positive for one or more of the three test organisms. The minimum number of bacterial genomic equivalents present in the average culture-negative ear was estimated to be greater than 10(4) based on dilutional experiments. CONCLUSIONS: The PCR-based assay systems can detect the presence of bacterial DNA in a significant percentage of culturally sterile middle ear effusions. While this finding is not proof of an active bacterial infectious process, the large number of bacterial genomic equivalents present in the ears is suggestive of an active process.
Bacteriological Techniques↗