Comparison of results generated by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence PCR used to characterize penicillin-resistant pneumococci from the United States.

One hundred forty-seven isolates of Streptococcus pneumoniae with high-level penicillin resistance collected during a national surveillance program in the United States were characterized by serotyping, pulsed-field restriction analysis, ribotyping, and repetitive-sequence (BOX element) PCR. The results generated by each method were compared by frequency of association to examine whether relationships existed between the various typing methods and statistically to determine association with the geographic source of the isolate or the age of the patient from whom the isolate was obtained. When the data were examined by pairwise analysis of individual strain classifications produced by each typing method, no statistically significant relationships between strain type, geographic location, or patient age were identified, suggesting that distinct clones of penicillin-resistant S. pneumoniae have been widely distributed throughout the United States. However, we did observed shared expression of two or three typing markers at a high frequency (>50%) among clusters of strains, indicating a certain level of concordance between the various typing methods used to classify penicillin-resistant S. pneumoniae.

Computed tomography vs. magnetic resonance imaging of acute bacterial sinusitis: a rabbit model.

PURPOSE: Computed topography (CT) and magnetic resonance imaging (MRI) are important, both clinically and in a research setting, in assessing bacterial sinusitis (BS). The use of CT scanning to evaluate sinus opacification in a reversible model of rabbit acute sinusitis has been reported. MRI offers the potential for better visualization of soft tissue and fluid changes within the paranasal sinuses. MRI has potential as a research tool in animal models of sinusitis. This article compares the use of CT and MRI in measuring maxillary sinus opacification in rabbits during experimental, reversible BS. MATERIALS AND METHODS: In 2 independent trials, New Zealand White rabbits were imaged for baseline anatomy, and BS was generated by sinus inoculation with Staphylococcus aureus. Serial imaging was performed as a measure of the progression and resolution of BS during the trials. Two experienced, independent reviewers then scored each CT and MRI for percent opacification of the maxillary sinus. These scores were analyzed to assess the degree of agreement between the reviewers. RESULTS: The correlation coefficients for CT and MRI were 0.6816 and 0.3584, respectively. The Z-statistic comparing these correlation coefficients was significant (P < .0001), indicating that CT is a more precise measure of reversible BS in this rabbit model. Differences in mean scan time and cost per scan were also significantly different (P < .0001), with CT being both quicker and less expensive. CONCLUSIONS: Greater interobserver consistency of scan interpretation, with less time and cost, make CT the preferred tool for measuring BS in this rabbit model. Attributes of MRI such as better resolution of fluid-tissue interfaces and custom surface coil design for visualization of specific anatomic structures are discussed as they may increase the effectiveness of MRI as an imaging modality in future sinusitis research.

Interleukin-1 receptor antagonist as an adjunct in the treatment of Haemophilus influenzae otitis media in the chinchilla.

OBJECTIVES: This purpose of this study was to investigate the effect of blockade of the inflammatory cytokine pathway on experimentally induced otitis media in the chinchilla model. STUDY DESIGN: Pilot, randomized placebo-controlled trial. METHODS: Ampicillin-sensitive Haemophilus influenzae otitis media was induced in 45 adult chinchillas. The animals were randomly assigned to the following treatment groups: 1) transbullar injections (TBI) of interleukin-1 receptor antagonist (IL-1ra) and intramuscular ampicillin, 2) TBI of saline and intramuscular ampicillin, 3) TBI of IL-1ra and intramuscular sa-1 line or 4) TBI of saline and intramuscular saline. Blinded investigators measured resolution of otitis media by otomicroscopy, tympanogram, and culture results. RESULTS: Comparisons were made between the treatment groups to assess the ability of IL-1ra to assist with resolution of otitis media using exact two-group binomial tests with the StatXact statistical program. The group with TBI of IL-1ra and intramuscular ampicillin as a treatment demonstrated trends suggesting more rapid resolution of positive cultures and more rapid and complete return to normal results on tympanograms and otomicroscopic findings compared with the group treated with TBI of saline and intramuscular ampicillin. These trends did not achieve statistical significance with the relatively small sample sizes used in this pilot study. CONCLUSIONS: This investigation provides further evidence that the inflammatory cytokine cascade plays a significant role in the pathophysiology of otitis media and that modulation of this inflammatory pathway may provide novel and efficacious treatments for otitis media Further studies with larger groups of animals are warranted to determine whether the trends identified in this pilot study are reproducible and achieve statistical significance.

Clonal relationships among high-level penicillin-resistant Streptococcus pneumoniae in the United States.

During the past 6 to 7 years, the problem of antimicrobial resistance in Streptococcus pneumoniae has grown dramatically in the United States. Currently, approximately 26.5% of pneumococcal isolates express intermediate levels of resistance to penicillin; approximately 17.5% are highly penicillin resistant. We studied whether clonal relationships exist among current isolates of high-level penicillin-resistant S. pneumoniae (PRSP) in the United States. One hundred forty-seven PRSP isolates recovered in a 30-center surveillance study in the United States during 1994-1995 were characterized with respect to serotype, antimicrobial susceptibility pattern, and pulsed-field gel electrophoresis (PFGE) profile. Only six serotypes were observed among the 147 PRSP isolates examined in this study: 6A, 6B, 9A, 14, 19F, and 23F. One hundred three (70.1%) of the 147 strains were characterized by one of only nine PFGE types; 76 (51.7%) of the 147 isolates were characterized by only four PFGE profiles. Currently in the United States, most PRSP strains are represented by relatively few clonal groups.

Evaluation of the premier EHEC assay for detection of Shiga toxin-producing Escherichia coli.

An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H-, and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.

Cryptosporidiosis in children during a massive waterborne outbreak in Milwaukee, Wisconsin: clinical, laboratory and epidemiologic findings.

During the spring of 1993 an estimated 403000 residents of the greater Milwaukee, Wisconsin area experienced gastrointestinal illness due to infection with the parasite Cryptosporidium parvum following contamination of the city's water supply. To define the clinical, laboratory and epidemiologic features of outbreak-associated cryptosporidiosis in children, medical and laboratory records for all children submitting stool samples to the microbiology laboratory of the Children's Hospital of Wisconsin between 7 April and 13 May 1993 were reviewed retrospectively. Interviews with parents were also conducted to obtain additional clinical history. Cryptosporidium, as the sole pathogen, was identified in stools from 49 (23%) of the 209 children enrolled in the study. Children with laboratory-confirmed cryptosporidiosis were more likely to live in areas of Milwaukee supplied with contaminated water (RR = 1.92, CI = 1.19-3.09), to be tested later in their illness (P < 0.05), to have submitted more than one stool specimen (P = 0.01), to have an underlying disease that altered their immune status (RR = 2.78, CI = 1.60-4.84), and to be older than 1 year of age (RR = 2.02, CI = 1.13-3.60). Clinical illness in these patients was more prolonged and associated with weight loss and abdominal cramps compared with Cryptosporidium-negative children. In the context of this massive waterborne outbreak relatively few children had documented infection with Cryptosporidium. If many children who tested negative for the parasite were truly infected, as the epidemiologic data suggest, existing laboratory tests for Cryptosporidium were insensitive, particularly early in the course of illness.

Antimicrobial effect on rabbit sinusitis after temporary ostial occlusion.

A key precipitating factor in the pathogenesis of acute bacterial sinusitis (ABS) is ostiomeatal obstruction. It has been postulated that ABS cannot resolve until the sinus ostium is patent. The primary purpose of this study is to quantify sinusitis resolution after temporary ostial occlusion in a rabbit model. A secondary aim is to quantify bacterial clearance with and without antimicrobial therapy in Staphylococcus aureus infected sinuses. A blinded, placebo controlled study in 10 New Zealand white rabbits was performed using Merocel as a temporary maxillary sinus ostial obstruction. Computerized tomography (CT) of the maxillary sinuses was obtained before and after bilateral ostial occlusion; sinuses were cultured and then the left sinus was injected with approximately 10(8) cfu/mL S. aureus. CT imaging and sinus cultures were obtained and then the ostial plugs were removed. Serial CT and cultures were done until sinusitis resolved and cultures were negative for S. aureus. Mean CT sinusitis scores increased with ostial obstruction and decreased with ostial plug removal (P < 0.0001 and P < 0.001). Injection of S. aureus did not significantly change mean CT sinusitis scores despite being cultured from 45% of sinuses. Antimicrobial therapy was not significant in reducing mean CT scan sinusitis scores or positive cultures. Resolution of mucosal swelling is more dependent on relief of ostial obstruction rather than antimicrobial therapy in this rabbit model.

Prevalence of human herpesvirus 6 in lung tissue from children with pneumonitis.

AIMS/BACKGROUND: Human herpesvirus type 6 (HHV-6) is the aetiological agent of exanthem subitum, and has also been linked with a variety of other diseases. The aim of this study was to investigate the role of HHV-6 in pneumonitis in children. METHODS: Formalin fixed, paraffin wax embedded lung tissue from 33 children (age range two months to 16 years) who died with pneumonitis was subjected to immunohistochemical staining for HHV-6 using an avidin-biotin method. RESULTS: Active HHV-6 infection was demonstrated in four children: a bone marrow transplant recipient with concomitant adenovirus infection, a patient with hepatitis of unknown aetiology, a patient with congenital anomalies, and a patient with congenital immunodeficiency. CONCLUSION: Accurate localisation of HHV-6 is possible in postmortem lung tissue. HHV-6 either alone or in combination with other pathogens may play a role in the development of pneumonitis.

Microbiological tolerance in orthopaedic infections: delayed response of septic arthritis and osteomyelitis of the hip due to infection with tolerant Staphylococcus aureus.

A 14-year-old boy presented to another hospital with a clinical picture of septic arthritis. After aspiration of purulent material from the joint, empiric antibiotic treatment was initiated and an arthrotomy was performed. Antibiotic treatment was then modified to nafcillin according to microbiological sensitivity results of the isolated Staphylococcus aureus as determined by minimal inhibitory concentration testing. One week later purulent drainage recurred and open drainage had to be repeated; an abscess anterior to the joint was noted. Once again the infection failed to resolve, and the patient was transferred to our institution where a third arthrotomy had to be performed. The organism isolated at the first aspiration was reexamined and found to have a minimal bactericidal concentration to minimal inhibitory concentration ratio of 32, implying a tolerant organism. The antibiotic treatment was modified to an antibiotic not subject to the tolerance phenomenon, and the infection resolved without additional surgical intervention.

Comparison of four different methods for detection of Cryptosporidium species.

Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost. We conclude that all methods tested were equally sensitive and specific for the detection of Cryptosporidium spp. Ease of use, adaptability to batch testing, and cost are important criteria in determining the method of choice.

Antigenic variation among Borrelia spp. in relapsing fever.

Seven antigens of Borrelia hermsii, B. parkeri, and B. turicatae with isoelectric points in the range of 4.4 to 5.0 and molecular masses of 40 to 43 kilodaltons played a role in the relapse phenomenon of relapsing fever. Based upon location of the antigens in the outer envelope, the molecular weight, and Western blot analysis, the antigens from each phase of spirochetemia appeared to be a mixture of the serotype-specific antigens of cloned B. hermsii.