Chronic high Epstein-Barr viral load state and risk for late-onset posttransplant lymphoproliferative disease/lymphoma in children.

Increased use of serial EBV-PCR monitoring after pediatric transplantation has led to the identification of asymptomatic patients who carry very high viral loads over prolonged periods. The significance of this high-load state is unknown. We speculated that this state may identify patients at high risk for development of late PTLD/lymphoma. We reviewed data on 71 pediatric heart recipients who had serial viral load monitoring since 1997. Chronic high-load state was defined as the presence of >16,000 genome copies/mL whole blood on > or =50% of samples over at least 6 months. Among 20 high-load carriers (eight following prior PTLD, seven with prior symptomatic EBV infection, five without previous EBV disease), 9 (45%) developed late-onset PTLD 2.5-8.4 years posttransplant (including with four Burkitt's lymphoma). Among 51 controls with low (n = 39) or absent (n = 12) loads, only 2 (4%; p < 0.001 absent/low vs. high load) developed late PTLD/lymphoma. By multivariable analysis, high-load carrier state (OR = 12.4, 95% CI 2.1-74.4) and prior history of PTLD (OR = 10.7, 95% CI 1.9-60.6) independently predicted late PTLD. A chronic high EBV-load state is not benign and is a predictor of de novo or recurrent PTLD.

Effect of a Mycoplasma hominis-like Mycoplasma on the infection of HEp-2 cells by the TW-183 strain of Chlamydia pneumoniae.

We isolated a Mycoplasma hominis-like mycoplasma from a stock culture of Chlamydia pneumoniae TW-183 obtained from the American Type Culture Collection and eradicated the contaminant by treating the stock suspension with a nonionic detergent, Igepal CA-630. The M. hominis-like mycoplasma neither inhibits nor enhances the infectivity of C. pneumoniae for HEp-2 cells.

Comparative evaluation of culture and PCR for the detection and determination of persistence of bacterial strains and DNAs in the Chinchilla laniger model of otitis media.

This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.

Efficacy of thermal treatment and copper-silver ionization for controlling Legionella pneumophila in high-volume hot water plumbing systems in hospitals.

BACKGROUND: Thermal treatment and copper-silver ionization are often used for controlling Legionella pneumophila in high-volume hospital plumbing systems, although the comparative efficacies of these measures in high-volume systems are unknown. METHODS: Thermal treatment of a hot water circuit was accomplished by flushing hot water (> 60 degrees C) through distal fixtures for 10 minutes. Copper-silver ionization was conducted in three circuits by installing units into return lines immediately upstream from hot water tanks. Recovery rates of L. pneumophila were monitored by culturing swab samples from faucets. Concentrations of copper and silver in water samples were determined by atomic absorption spectrophotometry. RESULTS: Four heat-flush treatments failed to provide long-term control of L. pneumophila. In contrast, ionization treatment reduced the rate of recovery of L. pneumophila from 108 faucets from 72% to 2% within 1 month and maintained effective control for at least 22 months. Only three samples (1.9%) of hot water from faucets exceeded Environmental Protection Agency standards for silver, and none exceeded the standards for copper. Of 24 samples obtained from hot water tanks, 42% and 50% exceeded the silver and copper standards, respectively. CONCLUSIONS: Copper-silver ionization effectively controls L. pneumophila in high-volume plumbing systems and is superior to thermal treatment; however, high concentrations of copper and silver can accumulate at the bottom of hot water tanks.

Multiplex PCR-based assay for detection of Bordetella pertussis in nasopharyngeal swab specimens.

A multiplex PCR-based assay was developed for the detection of Bordetella pertussis in nasopharyngeal swab specimens. The assay simultaneously amplified two separate DNA targets (153 and 203 bp) within a B. pertussis repetitive element and a 438-bp target within the beta-actin gene of human DNA (PCR amplification control). PCR products were detected by a sensitive and specific liquid hybridization gel retardation assay. A total of 496 paired nasopharyngeal swab specimens were tested by both the PCR-based assay and culture. Although 30 (6%) of the specimens inhibited the amplification of the beta-actin target, in all 29 specimens studied, the inhibition disappeared on repeat testing or was easily overcome with a 1:8 dilution or less of specimen digest. Of the 495 specimen pairs yielding a final evaluable result by the PCR-based assay, 19.0% were positive by the PCR-based assay, whereas 13.9% were positive by culture (P < 0.0001). After resolving the PCR-positive, culture-negative results by testing an additional aliquot from these specimens by the multiplex PCR-based assay, the PCR-based assay had a sensitivity and specificity of 98.9 and 99.7%, respectively, compared with values of 73.4 and 100%, respectively, for culture. In comparison with patients with culture-confirmed pertussis, those with PCR-positive, culture-negative results were older and more likely to have had prolonged cough, immunization with pertussis vaccine, or treatment with erythromycin. This multiplex PCR-based assay is substantially more sensitive than culture and identifies specimens that contain inhibitors of PCR.

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.

In vitro susceptibility of aural isolates of Pseudomonas aeruginosa to commonly used ototopical antibiotics.

The choice of antimicrobial agents used to treat Pseudomonas aeruginosa infections of the ear is quite empiric. Yet in spite of this, very little has been published examining susceptibility patterns of aural isolates of P. aeruginosa. Recently, increasing concern has emerged over the development of resistance to many of the commonly used ototopical preparations with activity against P. aeruginosa. This concern stems from the fact that these preparations have been in use for a long time, and P. aeruginosa is known to develop resistance fairly readily. We prospectively studied the susceptibilities of aural isolates of P. aeruginosa in 231 consecutive children who were seen in the outpatient Pediatric Otolaryngology Department at Children's Hospital of Pittsburgh during the years 1992 and 1993. The agents tested included neomycin, polymyxin B, colistin, and norfloxacin. We found that only 17.8% of the isolates were sensitive to neomycin, as opposed to > 95% for each of the other agents tested (polymyxin B, 99.6%; colistin, 97.4%; and norfloxacin, 98.3%). This difference proved to be statistically significant (p < 0.05). Given the concern of aminoglycoside-induced ototoxicity and the high rate of neomycin resistance, we believe that further investigation of other alternative ototopic agents with activity against P. aeruginosa is warranted.

Field inversion gel electrophoretic analysis of Legionella pneumophila strains associated with nosocomial legionellosis in children.

Two nosocomial cases of Legionnaires' disease occurred in children. Legionella pneumophila serogroup 1 was isolated from both patients and 30 of 39 plumbing system sites in the hospital. The patient and hospital environmental isolates yielded identical field inversion gel electrophoretic patterns which differed from patterns observed with epidemiologically unrelated strains.

Gentamicin-Containing Peptone-Yeast Extract Medium for Cocultivation of Hartmannella vermiformis ATCC 50256 and Virulent Strains of Legionella pneumophila.

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.

Therapeutic implications in the treatment of aural Pseudomonas infections based on in vitro susceptibility patterns.

OBJECTIVE: To examine the in vitro susceptibility patterns of aural isolates of Pseudomonas aeruginosa and to identify changes over a 4-year period. DESIGN: Retrospective case series. SETTING: The outpatient department at Children's Hospital of Pittsburgh (Pa), a tertiary referral center. PATIENTS: Ambulatory children younger than 18 years from whose ears P aeruginosa was isolated. OUTCOME MEASURES: The in vitro susceptibility of aural isolates of P aeruginosa to ampicillin, cefotaxime, chloramphenicol, sulfisoxazole, ticarcillin, mezlocillin, gentamicin, tobramycin, cefazolin, tetracycline, piperacillin, nitrofurantoin, cephalexin hydrochloride, ceftriaxone, cefuroxime axetil, and sulfamethoxazole-trimethoprim. RESULTS AND CONCLUSIONS: No changes were found in the trends of the susceptibility patterns over the 4-year study period, with the exception of the semisynthetic penicillins, ticarcillin and mezlocillin. These two agents were found to be relatively ineffective against the strains of P aeruginosa isolated in 1989 (59% and 18% susceptibility, respectively). This finding is in contrast to their effectiveness over the remainder of the study period (96% and 90% susceptibility, respectively), which was excellent. These observations likely reflect a change in the breakpoints for the minimal inhibitory concentrations between these periods. The intravenous agent with the best susceptibility profile was piperacillin (96%). Of the aminoglycosides tested, 94% of the isolates were sensitive to tobramycin, as opposed to only 79% for gentamicin. This finding may have significance when one is empirically selecting ototopical therapy, since both tobramycin and gentamicin are available as topical preparations. Of the oral agents, the combination of sulfamethoxazole-trimethoprim was most effective (46%).

Evaluation of airborne particulates and fungi during hospital renovation.

This study was conducted over 30 weeks on a hospital floor undergoing partial renovation. Some patients housed on the floor were immunosuppressed, including bone marrow transplant recipients. The construction zone was placed under negative pressure and was separated from patient rooms by existing hospital walls and via erection of a temporary barrier. Other control measures minimized patient exposure to airborne materials. Air sampling was done for 3 weeks prior to construction, 24 weeks during construction, and 3 weeks after renovation was completed. Airborne particulate concentrations, total spore counts, particle size, and fungal species were assessed. At the beginning of the renovation there were increases in airborne particulates (from 0.2 to 2.0 mg/m3) and fungal spores (from 3.5 to 350 colony forming units (CFU/m3), but only in the construction zone. Throughout the remainder of the renovation, particulate and fungal spore levels fluctuated inside the construction zone but remained close to baseline values in the patient area. When renovation was completed, particulates and spore counts inside the construction zone decreased to preconstruction levels. The primary fungus isolated from air samples was Penicillium. This study demonstrated that control measures were effective in reducing exposures of hospitalized patients to airborne particulates and spores and in reducing the increased risk of aspergillosis and other fungal infections associated with hospital construction projects. The data from this study may be useful in establishing exposure guidelines for other health care settings.

Identification of a patient with Streptococcus pneumoniae bacteremia and meningitis by the polymerase chain reaction (PCR).

A polymerase chain reaction (PCR) assay based on the penicillin-binding protein gene PBP2B identified the presence of DNA specific for Streptococcus pneumoniae in the serum and CSF of a patient with culture-proven bacteremia and meningitis. Positive signals were seen to dilutions of 1:125 and 1:390,625 for the blood and CSF specimens, respectively. Potential advantages of PCR over conventional culture include exquisite sensitivity, faster results and the ability to identify the organisms by the presence of species-specific DNA even in patients pretreated with antibiotics.

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.

Effect of experimental influenza A virus infection on isolation of Streptococcus pneumoniae and other aerobic bacteria from the oropharynges of allergic and nonallergic adult subjects.

Intranasal challenge with both influenza A virus and Streptococcus pneumoniae promotes otitis media with S. pneumoniae in chinchillas. We investigated whether influenza A virus infection promotes oropharyngeal colonization with S. pneumoniae and other middle ear pathogens by selectively inhibiting commensal bacteria. On study day 0, 12 allergic and 15 nonallergic adult subjects were intranasally inoculated with influenza A/Kawasaki (H1N1) virus. Every subject was infected with the virus as demonstrated by nasal shedding or seroconversion. Average upper respiratory symptom scores and nasal secretion weights from the entire subject group were elevated between days 2 and 6 (acute phase) and were not significantly different between allergic and nonallergic subjects. S. pneumoniae was not isolated from any subject prior to the virus challenge but was isolated in heavy density from 4 (15%) subjects on day 6 (P = 0.055). Staphylococcus aureus was isolated more frequently from the nonallergic subjects than from the allergic subjects on days 2 (80 versus 25%, respectively) 4, (67 versus 17%, respectively), and 6 (73 versus 25%, respectively) (P < 0.05). The isolation rates of other middle ear pathogens were not significantly different before virus challenge and during the acute and resolution phases (days 27 to 30) of the experimental infection for the entire subject group or either the allergic or nonallergic subgroup. Densities and isolation rates of commensal bacteria from the entire subject group were similar throughout the observational period. These results suggest that the virus infection promoted S. pneumoniae colonization of the oropharynx and that nonallergic persons may be more vulnerable to colonization with S. aureus than allergic persons. The altered colonization rates were not attributed to inhibition of commensal bacteria.

Detection of Streptococcus pneumoniae in whole blood by PCR.

Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole blood was developed. With this protocol it was possible to detect S. pneumoniae-specific DNA from whole blood specimens inoculated with as little as 4 CFU/ml. Copurified human blood DNA, ranging from 0 to 4.5 micrograms per PCR, did not affect the sensitivity of S. pneumoniae detection by PCR. A blinded clinical trial was used to compare the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained from pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh. With culture as the "gold standard," the PCR-based assay had a sensitivity of 80% (4 of 5 culture-positive specimens were PCR positive) and a specificity of 84% (26 of 31 culture-negative specimens were PCR negative). However, three patients whose specimens were PCR positive and culture negative had histories suggestive of bacteremia, including recent positive blood cultures, treatment with antibiotics, cellulitis, and multiple emergency room visits for fever within a 24-h period. These data suggest that PCR-based assays for S. pneumoniae may prove useful to augment current methods of detection for S. pneumoniae bacteremia.

Simple test of synergy between ampicillin and vancomycin for resistant strains of Enterococcus faecium.

The combination of ampicillin and vancomycin kills some but not all strains of ampicillin- and vancomycin-resistant Enterococcus faecium. We compared a simple test for synergy utilizing a commercially available microdilution susceptibility system with time-kill studies and determined acceptable breakpoints for this test for 20 strains of ampicillin- and vancomycin-resistant E. faecium. The combination of ampicillin and vancomycin was tested for synergy by time-kill, broth macrodilution, and broth microdilution procedures. Repeat testing of isolates by macro- and microdilution synergy methods yielded MICs that were within one twofold dilution of each other for both intra- and intertest comparisons. Synergy was always detected by time-kill studies when the MIC of ampicillin in the combination synergy screen was < or = 8 micrograms/ml in the presence of vancomycin. No synergy was detected when the MIC was > 16 micrograms/ml in the combination microdilution synergy screen. The determination of the synergy by the broth microdilution procedure appears to be simple, convenient, and accurate.

Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab.

A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 10(3) CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays.

Impact of Chlorine and Heat on the Survival of Hartmannella vermiformis and Subsequent Growth of Legionella pneumophila.

Hartmannella vermiformis, a common amoebal inhabitant of potable-water systems, supports intracellular multiplication of Legionella pneumophila and is probably important in the transportation and amplification of legionellae within these systems. To provide a practical guide for decontamination of potable-water systems, we assessed the chlorine and heat resistance of H. vermiformis. H. vermiformis cysts and trophozoites were treated independently with chlorine at concentrations of 2.0 to 10.0 ppm for 30 min and then cocultured with L. pneumophila. Both cysts and trophozoites were sensitive to concentrations between 2.0 and 4.0 ppm and above (trophozoites somewhat more so than cysts), and 10.0 ppm was lethal to both forms. Hartmannellae treated with chlorine up to a concentration of 4.0 ppm supported the growth of legionellae. To determine whether heat would be an effective addendum to chlorine treatment of amoebae, hartmannellae were subjected to temperatures of 55 and 60 degrees C for 30 min and alternatively to 50 degrees C followed by treatment with chlorine at a concentration of 2 ppm. Fewer than 0.05% of the amoebae survived treatment at 55 degrees C, and there were no survivors at 60 degrees C. Pretreatment at 50 degrees C appeared to make hartmannella cysts more susceptible to chlorine but did not further reduce the concentration of trophozoites.

Selective decontamination in pediatric liver transplants. A randomized prospective study.

Although it has been suggested that selective decontamination of the digestive tract (SDD) decreases postoperative aerobic Gram-negative and fungal infections in orthotopic liver transplantation (OLT), no controlled trials exist in pediatric patients. This prospective, randomized controlled study of 36 pediatric OLT patients examines the effect of short-term SDD on postoperative infection and digestive tract flora. Patients were randomized into two groups. The control group received perioperative parenteral antibiotics only. The SDD group received in addition polymyxin E, tobramycin, and amphotericin B enterally and by oropharyngeal swab postoperatively until oral intake was tolerated (6 +/- 4 days). Indications for operation, preoperative status, age, and intensive care unit and hospital length of stay were no different in SDD (n = 18) and control (n = 18) groups. A total of 14 Gram-negative infections (intraabdominal abscess 7, septicemia 5, pneumonia 1, urinary tract 1) developed in the 36 patients studied. Mortality was not significantly different in the two groups. However, there were significantly fewer patients with Gram-negative infections in the SDD group: 3/18 patients (11%) vs. 11/18 patients (50%) in the control group, P < 0.001. There was also significant reduction in aerobic Gram-negative flora in the stool and pharynx in patients receiving SDD. Gram-positive and anaerobic organisms were unaffected. We conclude that short-term postoperative SDD significantly reduces Gram-negative infections in pediatric OLT patients.