Steven D. Dallas

Binding of Clostridium difficile toxin A to human milk secretory component

Toxigenic Clostridium difficile is isolated from a majority of healthy human infants. The exact mechanism of asymptomatic colonisation is unclear; however, previous studies in this laboratory have shown that components of both the immunoglobulin and non-immunoglobulin fractions of human milk bind to toxin A and prevent its interaction with hamster intestinal brush border membranes (BBMs). Secretory IgA (sIgA) is the primary immunoglobulin found in human milk. As sIgA resists digestion in the infant stomach and passes at high levels into the colon, its ability to bind toxin A was the subject of this investigation. Purified sIgA in concentrations at and below those found in human milk inhibited the binding of toxin A to purified BBM receptors. Heating sIgA to 100 degrees C for 5 min did not affect its inhibitory activity. IgM, IgG and serum IgA did not appreciably inhibit the binding of toxin A to BBM receptors. SDS-PAGE separated sIgA into three major bands: secretory component, heavy chains and light chains. Autoradiography with radiolabelled toxin A revealed that toxin A bound to the secretory component (SC) of sIgA. When the three purified subunits of sIgA were coated on to microtitration wells, SC bound significantly more toxin A than the heavy or light chains of sIgA. Purified SC also inhibited toxin binding to receptors in a dose-dependent fashion similar to sIgA. The heavy and light chains of sIgA did not inhibit toxin A receptor binding. Removing carbohydrates from sIgA and SC by enzymic digestion showed that toxin A binds much less to deglycosylated SC than to glycosylated SC. These data suggest that SC in human milk binds to toxin A and may function as a receptor analogue, protecting human infants against C. difficile-associated disease.

Treatment of streptococcal pharyngitis with once-daily compared with twice-daily amoxicillin : A noninferiority trial

Background: Two relatively small previous studies comparing once-daily amoxicillin with conventional therapy for group A streptococcal (GAS) pharyngitis reported similar rates of bacteriologic success for each treatment group. The purpose of this study was to further evaluate once-daily amoxicillin for GAS pharyngitis in a larger study. Methods: In a single pediatric practice, from October through May for 2 consecutive years (2001–2003), we recruited children 3 to 18 years of age who had symptoms and signs suggestive of GAS pharyngitis. Patients with a positive rapid test for GAS were stratified by weight (<40 kg or ≥40 kg) and then randomly assigned to receive once-daily (750 mg or 1000 mg) or twice-daily (2 doses of 375 mg or 500 mg) amoxicillin for 10 days. We determined bacteriologic failure rates for GAS in the pharynx from subsequent swabs taken at 14 to 21 (visit 2) and 28 to 35 (visit 3) days after treatment initiation. We conducted a randomized, controlled, investigator-blinded, noninferiority trial to evaluate whether amoxicillin given once daily would have a bacteriologic failure rate no worse than that of amoxicillin given twice daily within a prespecified margin of 10%. GAS isolates were characterized to distinguish bacteriologic failures from new acquisitions. Adverse events were described and adherence was evaluated by review of returned daily logs and dosage bottles. Results: Of 2139 potential study patients during the 2-year period, we enrolled 652 patients, 326 into each treatment group. Children in the 2 groups were comparable with respect to all demographic and clinical characteristics except that children <40 kg more often presented with rash in each treatment group. At visit 2, failure rates were 20.1% (59 of 294) for the once-daily group and 15.5% (46 of 296) for the twice-daily group (difference, 4.53%; 90% confidence interval [CI], −0.6 to 9.7). At visit 3, failure rates were 2.8% (6 of 216) for the once-daily group and 7.1% (16 of 225) for the twice-daily group (difference, −4.33; 90% CI, −7.7 to −1.0). Gastrointestinal and other adverse events occurred in the once-daily treatment group with a frequency comparable to that in the twice-daily treatment group. Presumed allergic reactions occurred in 0.9% (6 of 635). More than 95% (516 of 541) of patients complied with 10 days of therapy with no significant differences between groups. Conclusions: We conclude that amoxicillin given once daily is not inferior to amoxicillin given twice daily. Gastrointestinal and other events did not occur significantly more often in the once-daily treatment group. From the data in this large, investigator-blinded, controlled study, once-daily amoxicillin appears to be a suitable regimen for treatment of GAS pharyngitis.

Treatment failure and costs in patients with methicillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections: A south Texas ambulatory research network (STARNet) study

Objective: To measure the incidence of treatment failure and associated costs in patients with methicillin-resistant Staphylococcus aureus skin and soft tissue infections (SSTIs). Methods: This was a prospective, observational study in 13 primary care clinics. Primary care providers collected clinical data, wound swabs, and 90-day follow-up information. Patients were considered to have “moderate or complicated” SSTIs if they had a lesion ≥5 cm in diameter or diabetes mellitus. Treatment failure was evaluated within 90 days of the initial visit. Cost estimates were obtained from federal sources. Results: Overall, treatment failure occurred in 21% of patients (21 of 98) at a mean additional cost of

Extrapharyngeal group A streptococcus infection: diagnostic accuracy and utility of rapid antigen testing

1,933.71 per patient. In a subgroup analysis of patients who received incision and drainage, those with moderate or complicated SSTIs had higher rates of treatment failure than those with mild or uncomplicated SSTIs (36% vs. 10%; P=.04). Conclusions: One in 5 patients presenting to a primary care clinic for a methicillin-resistant S. aureus SSTI will likely require additional interventions at an associated cost of almost

The automated clinical microbiology laboratory: fact or fantasy?

2,000 per patient. Baseline risk stratification and new treatment approaches are needed to reduce treatment failures and costs in the primary care setting.

The automated clinical microbiology laboratory

Background. Antigen tests have been well-studied and are widely used in pediatric practice for rapid detection of group A Streptococcus (GAS) infections in the throat, but they have not been examined sufficiently for the detection of infection of skin sites, such as the perineal region or impetiginous lesions. Methods. During the 3-year period 1999 to 2002, we evaluated 239 patients with suspected GAS skin infection, in 5 pediatric practices, using 3 Dacron swabs for each site. The first swab was tested in the pediatric office laboratory with an antigen detection kit. For the first 91 patients, the Abbott Test Pack Plus antigen detection test (ADT) was used. The Abbott Signify Strep A ADT was used to test subsequent patients. The second swab was tested with BD Directigen 1-2-3 ADT in the hospital laboratory. The third swab was placed in modified Stuart’s transport medium for comparison of recovery of GAS from culture in broth or on agar. A positive culture served as the reference standard. Test performance and test accuracy were determined for each ADT. Results. Of the 247 ADTs and cultures performed on 239 patients, 91 with suspected skin infection were tested with the Test Pack Plus test, 149 with the Signify Strep A test and 247 with the Directigen test. Eighty-six (35%) cultures were positive, 73 from perineal sites (54 rectal, 13 vaginal, 6 penile) and 13 from impetiginous lesions. There was 100% concordance for the 86 cultures positive for GAS in a comparison between dry Dacron swabs and swabs that had been placed in modified Stuart’s transport medium. Test Pack Plus and Signify Strep A ADTs had similar performance characteristics for skin infections: sensitivity, 92 and 88%; specificity, 99 and 97%; positive predictive value, 96 and 94%; and negative predictive value, 97 and 93%. Directigen ADT had sensitivity 78%, specificity 100%, positive predictive value 100% and negative predictive value 89%. Accuracy for the tests varied from 92 to 97%. Conclusion. Tests designed to detect GAS carbohydrate antigen in patients with pharyngitis can be used rapidly and accurately to detect GAS antigen in patients with cutaneous lesions suspected of GAS infection.

Development of Doxycycline MIC and Disk Diffusion Interpretive Breakpoints and Revision of Tetracycline Breakpoints for Streptococcus pneumoniae

ABSTRACT Automated chemistry laboratories dependent on robotic processes are the standard in both academic and large community hospital settings. Diagnostic microbiology manufacturers are betting that robotics will be used for specimen processing, plate reading, and organism identification in the near future. These systems are highly complex and have large footprints and hefty price tags. However, they are touted as being more efficient, rapid, and accurate than standard processes. Certain features, such as image collection, are highly innovative. Hospital administrators may be swayed to institute these new systems because of the promise of the need for fewer skilled workers, higher throughput, and greater efficiency. They also may be swayed by the fact that workers with the requisite clinical microbiology skills are becoming more difficult to find, and this technology should allow fewer skilled workers to handle larger numbers of cultures. In this Point-Counterpoint, Nate Ledeboer, Medical Director, Clinical Microbiology and Molecular Diagnostics, Dynacare Laboratories, and Froedtert Hospital, Milwaukee, WI, will explain why he believes that this approach will become widespread, while Steve Dallas of the University of Texas Health Science Center San Antonio explains why he thinks that this automation may not become widely used.

Comparative Whole Genome Sequencing of Community-Associated Methicillin-Resistant Staphylococcus aureus Sequence Type 8 from Primary Care Clinics in a Texas Community

ABSTRACT Automated chemistry laboratories dependent on robotic processes are the standard in both academic and large community hospital settings. Diagnostic microbiology manufacturers are betting that robotics will be used for specimen processing, plate reading, and organism identification in the near future. These systems are highly complex and have large footprints and hefty price tags. However, they are touted as being more efficient, rapid, and accurate than standard processes. Certain features, such as image collection, are highly innovative. Hospital administrators may be swayed to institute these new systems because of the promise of the need for fewer skilled workers, higher throughput, and greater efficiency. They also may be swayed by the fact that workers with the requisite clinical microbiology skills are becoming more difficult to find, and this technology should allow fewer skilled workers to handle larger numbers of cultures. In this Point-Counterpoint, Nate Ledeboer, Medical Director, Clinical Microbiology and Molecular Diagnostics, Dynacare Laboratories, and Froedtert Hospital, Milwaukee, WI, will explain why he believes that this approach will become widespread, while Steve Dallas of the University of Texas Health Science Center San Antonio explains why he thinks that this automation may not become widely used.

Predictors of community-associated Staphylococcus aureus, methicillin-resistant and methicillin-susceptible Staphylococcus aureus skin and soft tissue infections in primary-care settings.

ABSTRACT A study was performed to derive susceptibility testing interpretive breakpoints for doxycycline with Streptococcus pneumoniae and to reassess breakpoints for tetracycline using the requirements defined in Clinical and Laboratory Standards Institute (CLSI) document M23-A3. Tetracycline and doxycycline MICs and disk diffusion zone sizes were determined on 189 isolates selected from the 2009-2010 CDC Active Bacterial Core surveillance strain collection according to the testing methods described in CLSI documents M07-A8 and M02-A10. Tetracycline and doxycycline MICs and zones were compared to each other directly, and the reproducibility of MICs and zone diameters for both drugs was determined. Scattergrams of tetracycline MICs versus corresponding zone diameters and doxycycline MICs versus zones were prepared, and analysis indicated that the present CLSI tetracycline MIC and disk breakpoints did not fit the susceptibility data for doxycycline. Doxycycline was 1 to 3 dilutions more potent than tetracycline, especially in strains harboring the tetM resistance determinant. tetM was detected in ≥90% of isolates having tetracycline MICs of ≥4 μg/ml and in ≥90% with doxycycline MICs of ≥1. Limited pharmacokinetic/pharmacodynamic (PK/PD) data coupled with application of the error-rate bounded method of analysis suggested doxycycline-susceptible breakpoints of either ≤0.25 μg/ml or ≤0.5 μg/ml, with intermediate and resistant breakpoints 1 and 2 dilutions higher, respectively. The disk diffusion zone diameter correlates were susceptible at ≥28 mm, intermediate at 25 to 27 mm, and resistant at ≤24 mm. Revised lower tetracycline MIC breakpoints were suggested as susceptible at ≤1 μg/ml, intermediate at 2 μg/ml, and resistant at ≥4 μg/ml. Suggested tetracycline disk diffusion zones were identical to those of doxycycline.

Staphylococcus lugdunensis bacteremia and endocarditis treated with cefazolin and rifampin

Our understanding of the molecular dynamics driving the community‐associated methicillin‐resistant Staphylococcus aureus (CA‐MRSA) epidemic at the whole genome level is limited. We sought to assess the use of whole genome sequencing (WGS) to evaluate the genomic diversity and genotypic prediction of antimicrobial resistance of CA‐MRSA isolates from patients in South Texas.