Bobby L. Boyanton

DNA Pyrosequencing-Based Bacterial Pathogen Identification in a Pediatric Hospital Setting

ABSTRACT Sole reliance on biochemical methods can limit the clinical microbiology laboratorys ability to identify bacterial pathogens. This study describes the incorporation of DNA pyrosequencing-based identification for routine pathogen identification of atypical clinical isolates in a large childrens hospital. The assay capitalized on the highly conserved nature of 16S rRNA genes by positioning amplification and sequencing primers in conserved target sequences flanking the variable V1 and V3 regions. A total of 414 isolates of 312 pediatric patients were tested by DNA pyrosequencing during the time period from December 2003 to July 2006. Seventy-eight different genera were specified by DNA pyrosequencing, and isolates were derived from diverse specimen types. By integrating DNA sequencing of bacterial pathogens with conventional microbiologic methods, isolates that lacked a definitive identification by biochemical testing yielded genus- or species-level identifications in approximately 90% of cases by pyrosequencing. Improvements incorporated into the assay process during the period of clinical testing included software enhancements, improvements in sequencing reagents, and refinements in database search strategies. Coupled with isolation by bacteriologic culture and biochemical testing, DNA pyrosequencing-based bacterial identification was a valuable tool that markedly improved bacterial pathogen identification in a pediatric hospital setting.

Rapid Stool-Based Diagnosis of Clostridium difficile Infection by Real-Time PCR in a Children's Hospital

ABSTRACT Clostridium difficile is a major cause of nosocomial antibiotic-associated infectious diarrhea and pseudomembranous colitis. Detection of C. difficile by anaerobic bacterial culture and/or cytotoxicity assays has been largely replaced by rapid enzyme immunoassays (EIA). However, due to the lack of sensitivity of stool EIA, we developed a multiplex real-time PCR assay targeting the C. difficile toxin genes tcdA and tcdB. Stool samples from hospitalized pediatric patients suspected of having C. difficile-associated disease were prospectively cultured on cycloserine-cefoxitin-fructose agar following alcohol shock. Six testing modalities were evaluated, including stool EIA, culture EIA, and real-time PCR (tcdA and tcdB) of cultured isolates and stool samples. Real-time PCR detection was performed with tcdA and tcdB gene-specific primers and hydrolysis probes using the LightCycler platforms (Roche Diagnostics, Indianapolis, IN). A total of 157 samples from 96 pediatric patients were analyzed. The sensitivities of stool real-time PCR and stool EIA were 95% and 35%, respectively, with a specificity of 100% for both methods. The lower limit of detection of the stool real-time PCR was 30 CFU/ml of stool sample per reaction for tcdA and tcdB. This study highlights the poor performance of stool toxin EIAs in pediatric settings. Direct detection of C. difficile toxin genes in stool samples by real-time PCR showed sensitivity superior to that of stool and culture EIAs and performance comparable to that of real-time PCR assay of cultured isolates. Real-time PCR of DNA from stool samples is a rapid and cost-effective diagnostic modality for children that should facilitate appropriate patient management and halt the practice of serial testing by EIA.

Hansen disease in the United States in the 21st century: a review of the literature.

In this article we review the recent literature on Hansen disease (leprosy). We searched published literature through PubMed (National Library of Medicine) and extracted data through direct review of the literature and pathologic slides. Hansen disease continues to occur in the United States, including among the native-born population. Inclusion of the disease in the differential diagnosis is key to confirmation. Current epidemiology, classification systems, prevention measures, and therapy are reviewed.

Evaluation of the BD MAX GBS assay to detect Streptococcus group B in LIM broth–enriched antepartum vaginal–rectal specimens

The BD MAX GBS real-time polymerase chain reaction assay was evaluated concomitantly with Centers for Disease Control and Prevention-endorsed culture methods to detect Streptococcus group B from LIM broth-enriched antepartum vaginal-rectal specimens. The sensitivity of both methods exceeded 98%.

Evaluation of the Lyra Direct Strep Assay To Detect Group A Streptococcus and Group C and G Beta-Hemolytic Streptococcus from Pharyngeal Specimens

ABSTRACT The Lyra Direct strep assay was compared to culture for its ability to detect Streptococcus group A and β-hemolytic groups C/G using rapid antigen-negative pharyngeal specimens (n = 161). The Lyra assay correctly detected all β-hemolytic streptococci (group A, n = 19; group C/G, n = 5). In batch mode, the Lyra assay reduced intralaboratory turnaround time by 60% (18.1 h versus 45.0 h) but increased hands-on time by 96% (3 min 16 s versus 1 min 40 s per specimen).

Performance of the Affirm VP-III using residual vaginal discharge collected from the speculum to characterize vaginitis in symptomatic women.

Objective To evaluate the ability of collecting the Affirm VP-III test sample using the residual vaginal discharge found on the speculum. Methods and Methods One hundred nine symptomatic women (≥18 y) participated in this study. During pelvic examination, vaginal fluid was collected onto 3 swabs for office-based diagnostic tests and Affirm (referred to as Affirm-R). A fourth swab was used to collect residual vaginal discharge from the speculum, followed by Affirm testing (referred to as Affirm-RVD). Sensitivity, specificity, and Cohen &kgr; agreement for office-based diagnostic tests and Affirm-RVD were determined against Affirm-R. Results Complete results were available for 99 samples. Cohen &kgr; agreement between Affirm-RVD and Affirm-R was 0.66 (p < .0001) for Gardnerella vaginalis, 0.81 (p < .0001) for Candida species, and 1.0 (p < .0001) for Trichomonas vaginalis. Affirm-RVD sensitivity, specificity, and positive and negative predictive values were 73.8%, 91.2%, 86.1%, and 82.5% for G. vaginalis; 84.2%, 96.3%, 84.2%, and 96.3% for Candida species; and 100%, 100%, 100%, and 100% for T. vaginalis, respectively. Cohen &kgr; agreement between office-based diagnostic tests and Affirm-R was 0.16 (p = .141) for G. vaginalis, 0.46 (p < .0001) for Candida species, and 0.55 (p < .0001) for T. vaginalis. Conclusions The Affirm VP-III sample collected from the residual vaginal discharge found on the speculum after performing office-based diagnostic tests can produce comparable results to traditionally collected sample.