Charles W Stratton

Publications of Charles W Stratton

• Comparison of the Luminex xTAG RVP FAST and the Idaho Technology FilmArray RP Assays for the Detection of Respiratory viruses in Pediatric Patients at a Cancer Hospital.

  Authors: N Esther Babady, Peter Mead, Jeffrey Stiles, Carrie Brennan, Haijing Li, Susan Shuptar, Charles W Stratton, Yi-Wei Tang, Mini Kamboj

  Journal of clinical microbiology. 04/2012;

  Background: Respiratory viruses have become increasingly recognized as serious causes of morbidity and mortality in immunocompromised patients. Rapid and sensitive detection of respiratory viruses isBackground: Respiratory viruses have become increasingly recognized as serious causes of morbidity and mortality in immunocompromised patients. Rapid and sensitive detection of respiratory viruses is essential for early diagnosis and administration of appropriate antiviral therapy as well as for effective implementation of infection control measures.Methods: We compared the performance of two commercial assays, the xTAG RVP FAST (Luminex Diagnostics, Toronto, Canada) and the FilmArray RVP (FA RVP, Idaho Technology, Salt Lake City, Utah) in pediatric patients at Memorial Sloan-Kettering Cancer Center. These assays detect the following viruses: respiratory syncytial virus, influenza A and B, parainfluenza 1, 2, 3, 4, human metapneumovirus, adenovirus, enterovirus-rhinovirus, coronaviruses NL63, HKU1, 229E and OC43 and bocavirus. We tested a total of 358 respiratory specimens from 173 pediatric patients previously tested by direct fluorescence assay (DFA) and viral culture.Results: The overall detection rate (positive specimen/total specimen) for viruses tested by all methods was 24% for DFA/culture, 45% for xTAG RVP FAST and 51% for FA RVP. The agreement between the two multiplex assays was 84.5%, and the difference in detection rate was statistically significant (p<0.0001).Conclusion: Overall, the FA RVP was more sensitive than the xTAG RVP FAST and had a turn-around time of approximately 1 hour. The sensitivity, simplicity, and the random access platform make the FA RVP an excellent choice for the laboratory on-demand service with low to medium volume.

• High Proportion of Fluoroquinolone-Resistant M. tuberculosis Isolates with Novel Gyrase Polymorphisms and a gyrA Region Associated with Fluoroquinolone Susceptibility.

  Authors: Rose Devasia, Amondrea Blackman, Svetlana Eden, Haijing Li, Fernanda Maruri, Ayumi Shintani, Charles Alexander, Anne Kaiga, Charles W Stratton, Jon Warkentin, Yi-Wei Tang, Timothy R Sterling

  Journal of clinical microbiology. 12/2011;

  Fluoroquinolone resistance in M. tuberculosis can be conferred by mutations in gyrA or gyrB. The prevalence of resistance mutations outside the quinolone-resistance determining region (QRDR) of gyrAFluoroquinolone resistance in M. tuberculosis can be conferred by mutations in gyrA or gyrB. The prevalence of resistance mutations outside the quinolone-resistance determining region (QRDR) of gyrA or gyrB is unclear, since such regions are rarely sequenced. M. tuberculosis isolates from 1,111 patients with newly-diagnosed culture-confirmed tuberculosis diagnosed in Tennessee 2002-2009 were screened for phenotypic ofloxacin resistance (>2 μg/ml). For each resistant isolate, two ofloxacin-susceptible isolates were selected: one with antecedent fluoroquinolone exposure and one without. The complete gyrA and gyrB genes were sequenced and compared with M. tuberculosis H37Rv. Of 25 ofloxacin-resistant isolates, 11 (44%) did not have previously-reported resistance mutations, of which 10 had novel polymorphisms: 3 in the QRDR of gyrA, 1 in the QRDR of gyrB, and 6 outside the QRDR of gyrA or gyrB; 1 did not have any gyrase polymorphisms. Polymorphisms in gyrA codons 1-73 were more common in fluoroquinolone-susceptible than -resistant strains (20% vs. 0%; P = 0.016). In summary, almost half of fluoroquinolone-resistant M. tuberculosis isolates did not have previously described resistance mutations, which has implications for genotypic diagnostic tests.

• Comparative evaluation of Bruker Biotyper and BD Phoenix systems for identification of bacterial pathogens associated with urinary tract infections.

  Authors: Yingjun Yan, Shufang Meng, Dongmo Bian, Criziel Quinn, Haijing Li, Charles W Stratton, Yi-Wei Tang

  Journal of clinical microbiology. 09/2011; 49(11):3936-9.

  The Bruker Biotyper and BD Phoenix systems were evaluated for identification of 1,024 bacterial urinary tract isolates. The Biotyper and Phoenix systems correctly identified 99.9% and 99.5% to theThe Bruker Biotyper and BD Phoenix systems were evaluated for identification of 1,024 bacterial urinary tract isolates. The Biotyper and Phoenix systems correctly identified 99.9% and 99.5% to the genus level and 99.1% and 98.5% to the species level, respectively. Both systems provide reliable results, and the Biotyper system offers a rapid tool for urine bacterial isolate identification.

• Coccidioides, Cryptococcus, or Blastomyces? A Diagnostic Dilemma Encountered During Frozen Section Evaluation.

  Authors: Colt M McClain, Charles W Stratton, James D Chappell, Gerald T Van Horn

  Pediatric and developmental pathology : the official journal of the Society for Pediatric Pathology and the Paediatric Pathology Society. 08/2011;

  Abstract Intra-operative consultation via frozen section is an important part of modern day surgical pathology. Recognizing fungi in tissues on frozen and permanent sections is not always a simpleAbstract Intra-operative consultation via frozen section is an important part of modern day surgical pathology. Recognizing fungi in tissues on frozen and permanent sections is not always a simple task and correctly identifying the agent can be a significant challenge, even for experienced microscopists. We present a case of a 17 year old boy with chronic osteomyelitis involving the right proximal ulna. During an irrigation and debridement operation, a frozen section was sent to surgical pathology for evaluation. A limited patient history coupled with sparse organisms present in the frozen section led to the diagnosis of fungal osteomyelitis, favor Coccidioides. Follow-up permanent sections with special staining and successful fungal culture clarified the causal agent to be Blastomyces dermatitidis. The role of frozen sections is not to perfectly speciate the fungal pathogen, but to describe the morphology and infectious process and provide a differential of the candidate fungi. The importance of intra-operative culture in infectious cases cannot be understated, and it is the responsibility of pathologists to inform surgeons that tissue is needed for culture. A brief overview of Blastomyces, including histopathologic features and key microscopic differences from Coccidioides and Cryptococcus, is discussed. Key Words: Blastomyces, Coccidioides, Cryptococcus, Frozen Section, Fungus, Osteomyelitis.

• Management of immune dysfunction after adult cardiac surgery.

  Authors: J Scott Rankin, Olusola Oguntolu, Robert S Binford, D Scott Trochtenberg, Lawrence H Muhlbaier, Charles W Stratton

  The Journal of thoracic and cardiovascular surgery. 07/2011; 142(3):575-80.

  Pulmonary dysfunction/multiorgan failure syndrome is an important cause of mortality and morbidity after cardiac operations. In this series, results of immune augmentation were assessed in patientsPulmonary dysfunction/multiorgan failure syndrome is an important cause of mortality and morbidity after cardiac operations. In this series, results of immune augmentation were assessed in patients experiencing pulmonary dysfunction/multiorgan failure syndrome after cardiac surgery. Since 2002, 44 consecutive patients with primary antibiotic-refractory pulmonary dysfunction/multiorgan failure syndrome were treated with intravenous immunoglobulin (0.3 g/kg × 5 days; 1.5 g/kg total dose). Thirty patients had undergone complex valve or aortic surgery, and 14 patients had coronary bypass. Median age was 66 years, and risk profiles were especially high preoperatively. Clinical variables were assessed for 3 days prior (-3) to beginning intravenous immunoglobulin (on day 0) and for 5 days afterward (+5). A postoperative morbidity index was generated as a weighted sum of all relevant clinical variables. By using each patient as his or her own control, the therapeutic effect of intravenous immunoglobulin was assessed with linear regression of postoperative morbidity index over time with a spline and a knot at day 0, coincident with beginning intravenous immunoglobulin. At day 0, all patients were deteriorating clinically and refractory to major antibiotics. Overall morbidity was high, and immunoglobulin-G levels, obtained in the last 14 patients, were consistently low. By using linear regression of postoperative morbidity index over time, intravenous immunoglobulin administration was associated with significant improvement in clinical status (P < .0001). A total of 42 of 44 patients (95%) recovered uneventfully to hospital discharge. No significant complications of intravenous immunoglobulin therapy occurred. This experience suggests that management of immune dysfunction with intravenous immunoglobulin is safe and effective for treatment of primary pulmonary dysfunction/multiorgan failure syndrome after cardiac surgery. Expanded application seems indicated.

• Improved identification of yeast species directly from positive blood culture media by combining Sepsityper specimen processing and Microflex analysis with the matrix-assisted laser desorption ionization Biotyper system.

  Authors: Yingjun Yan, Ying He, Thomas Maier, Criziel Quinn, Gongyi Shi, Haijing Li, Charles W Stratton, Markus Kostrzewa, Yi-Wei Tang

  Journal of clinical microbiology. 05/2011; 49(7):2528-32.

  Current methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored theCurrent methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored the use of a matrix-assisted laser desorption ionization (MALDI) Biotyper system in combination with Sepsityper specimen processing and Microflex analysis for improved detection and identification of yeast species directly from positive blood culture specimens demonstrating yeast-like organisms by Gram stain. The limit of detection of yeast species in blood culture medium was determined to be 5.9 × 10(5) CFU, with intra- and interstrain coefficients of variation of 1.8 to 3.6% and 2.9%, respectively. A total of 42 yeast-containing positive blood culture specimens were processed, and the identification results were compared to those obtained by routinely used phenotypic methods. Specimens with discrepant results between the Biotyper and phenotypic methods were identified on the basis of internal transcribed spacer region sequencing. The MALDI Biotyper system correctly identified the 42 specimens to species level, including 28 (66.7%) Candida albicans, 8 (19.0%) Candida parapsilosis, and 5 (11.9%) Candida tropicalis isolates and 1 (2.4%) Cryptococcus neoformans isolate. The entire procedure, from specimen extraction to final result reporting, can be completed within 1 h. Our data indicated that the Sepsityper specimen processing and Microflex analysis by the MALDI Biotyper system provide a rapid and reliable tool for yeast species identification directly from positive blood culture media.

• Serum microRNA expression profile distinguishes enterovirus 71 and coxsackievirus 16 infections in patients with hand-foot-and-mouth disease.

  Authors: Lunbiao Cui, Yuhua Qi, Haijing Li, Yiyue Ge, Kangchen Zhao, Xian Qi, Xiling Guo, Zhiyang Shi, Minghao Zhou, Baoli Zhu, Yan Guo, Jun Li, Charles W Stratton, Yi-Wei Tang, Hua Wang

  PloS one. 01/2011; 6(11):e27071.

  Altered circulating microRNA (miRNA) profiles have been noted in patients with microbial infections. We compared host serum miRNA levels in patients with hand-foot-and-mouth disease (HFMD) caused byAltered circulating microRNA (miRNA) profiles have been noted in patients with microbial infections. We compared host serum miRNA levels in patients with hand-foot-and-mouth disease (HFMD) caused by enterovirus 71 (EV71) and coxsackievirus 16 (CVA16) as well as in other microbial infections and in healthy individuals. Among 664 different miRNAs analyzed using a miRNA array, 102 were up-regulated and 26 were down-regulated in sera of patients with enteroviral infections. Expression levels of ten candidate miRNAs were further evaluated by quantitative real-time PCR assays. A receiver operating characteristic (ROC) curve analysis revealed that six miRNAs (miR-148a, miR-143, miR-324-3p, miR-628-3p, miR-140-5p, and miR-362-3p) were able to discriminate patients with enterovirus infections from healthy controls with area under curve (AUC) values ranged from 0.828 to 0.934. The combined six miRNA using multiple logistic regression analysis provided not only a sensitivity of 97.1% and a specificity of 92.7% but also a unique profile that differentiated enterovirial infections from other microbial infections. Expression levels of five miRNAs (miR-148a, miR-143, miR-324-3p, miR-545, and miR-140-5p) were significantly increased in patients with CVA16 versus those with EV71 (p<0.05). Combination of miR-545, miR-324-3p, and miR-143 possessed a moderate ability to discrimination between CVA16 and EV71 with an AUC value of 0.761. These data indicate that sera from patients with different subtypes of enteroviral infection express unique miRNA profiles. Serum miRNA expression profiles may provide supplemental biomarkers for diagnosing and subtyping enteroviral HFMD infections.

• Simultaneous identification of mycobacterial isolates to the species level and determination of tuberculosis drug resistance by PCR followed by electrospray ionization mass spectrometry.

  Authors: Christian Massire, Cristina Agasino Ivy, Robert Lovari, Natalia Kurepina, Haijing Li, Lawrence B Blyn, Steven A Hofstadler, George Khechinashvili, Charles W Stratton, Rangarajan Sampath, Yi-Wei Tang, David J Ecker, Barry N Kreiswirth

  Journal of clinical microbiology. 12/2010; 49(3):908-17.

  Mycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used toMycobacterium tuberculosis that is resistant to both isoniazid (INH) and rifampin (RIF) is spreading. It has become a public health problem in part because the standard culture methods used to determine the appropriate treatment regimen for patients often take months following the presumptive diagnosis of tuberculosis. Furthermore, the misidentification of nontuberculosis mycobacteria (NTM) in patients presumably suffering from tuberculosis results in additional human and health care costs. The mechanisms of resistance for several drugs used to treat Mycobacterium tuberculosis are well understood and therefore should be amenable to determination by rapid molecular methods. We describe here the use of PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) in an assay that simultaneously determines INH and RIF resistance in Mycobacterium tuberculosis and identifies and determines the species of NTMs. The assay panel included 16 primer pairs in eight multiplexed reactions and was validated using a collection of 1,340 DNA samples from cultured specimens collected in the New York City area, the Republic of Georgia, and South Africa. Compared with phenotypic data, the PCR/ESI-MS assay had 89.3% sensitivity and 95.8% specificity in the determination of INH resistance and 96.3% sensitivity and 98.6% specificity in the determination of RIF resistance. Based on a set of 264 previously characterized liquid culture specimens, the PCR/ESI-MS method had 97.0% sensitivity and 99.9% specificity for determination of NTM identity. The assay also provides information on ethambutol, fluoroquinolone, and diarylquinoline resistance and lineage-specific polymorphisms, to yield highly discriminative digital signatures potentially suitable for epidemiology tracking.

• Assessment of Clostridium difficile infections by quantitative detection of tcdB toxin by use of a real-time cell analysis system.

  Authors: Alex B Ryder, Ying Huang, Haijing Li, Min Zheng, Xiaobo Wang, Charles W Stratton, Xiao Xu, Yi-Wei Tang

  Journal of clinical microbiology. 11/2010; 48(11):4129-34.

  We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells toWe explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin, a significant correlation between toxin protein concentrations and clinical CDI severities was observed (R(2) = 0.732, P = 0.0004). Toxin concentrations after treatment (0.89 ng/ml) were significantly lower than those prior to the treatment (15.68 ng/ml, Wilcoxon P = 0.01). The study demonstrates that the RTCA assay provides a functional tool for the potential assessment of C. difficile infections.

• Mass spectrometry biotyper system identifies enteric bacterial pathogens directly from colonies grown on selective stool culture media.

  Authors: Ying He, Haijing Li, Xuedong Lu, Charles W Stratton, Yi-Wei Tang

  Journal of clinical microbiology. 11/2010; 48(11):3888-92.

  We evaluated the performance and cost-effectiveness of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based Biotyper system for the routine identification of commonWe evaluated the performance and cost-effectiveness of a matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based Biotyper system for the routine identification of common enteric bacterial pathogens seen in middle Tennessee from suspicious colonies grown on selective stool culture media. A total of 304 suspicious colonies were selected and further identified from 605 stool specimens. The suspicious colonies were analyzed by the Biotyper system, and the results were compared to those from routine phenotypic methods, which identified 22 Salmonella species, 39 Shigella species, 3 enterohemorrhagic Escherichia coli (EHEC) isolates, 2 Yersinia enterocolitica isolates, 2 Campylobacter species, and 236 gastrointestinal normal flora isolates. The Biotyper system correctly identified the Salmonella species, Yersinia enterocolitica, and Campylobacter species but failed to distinguish the Shigella species and EHEC isolates from E. coli. Among the 236 normal flora isolates, 233 (98.7%) and 228 (96.6%) agreed at the genus and species levels, respectively, between the phenotypic and Biotyper methods. Organism identification scores were insignificantly different between colonies directly from selective media and subsequently from pure subculture. The entire Biotyper identification procedure, from smear preparation to final result reporting, can be completed within 30 min. The Biotyper system provides a rapid and simple screening tool for identifying many, but not all, suspicious colonies grown on selective media within 24 h after inoculation, which shortens test turnaround time by 2 to 3 days.

• Complex febrile seizures followed by complete recovery in an infant with high-titer 2009 pandemic influenza A (H1N1) virus infection.

  Authors: Mandy F O'Leary, James D Chappell, Charles W Stratton, Robert M Cronin, Mary B Taylor, Yi-Wei Tang

  Journal of clinical microbiology. 10/2010; 48(10):3803-5.

  We describe a 2009 H1N1 virus infection with a high viral load in a previously healthy infant who presented with complex febrile seizures and improved on oseltamivir without neurologic sequelae.We describe a 2009 H1N1 virus infection with a high viral load in a previously healthy infant who presented with complex febrile seizures and improved on oseltamivir without neurologic sequelae. Febrile seizures may be a complication in young children experiencing infection with high viral loads of 2009 H1N1 influenza virus.

• Characterization of three Staphylococcus aureus isolates from a 17-year-old female who died of tampon-related toxic shock syndrome.

  Authors: Yi-Wei Tang, Eric Himmelfarb, Marcia Wills, Charles W Stratton

  Journal of clinical microbiology. 03/2010; 48(5):1974-7.

  We report the identification and characterization of three Staphylococcus aureus isolates recovered from throat and vaginal cultures, as well as from an axillary abscess, of a 17-year-old female whoWe report the identification and characterization of three Staphylococcus aureus isolates recovered from throat and vaginal cultures, as well as from an axillary abscess, of a 17-year-old female who died of tampon-related toxic shock syndrome. The three S. aureus isolates were unrelated as determined by pulsed-field gel electrophoresis. The vaginal isolate was mecA, Panton-Valentine leukocidin, and staphylococcal enterotoxin B and C negative, toxic shock syndrome toxin 1 positive, and staphylococcal cassette chromosome mec element (SCCmec) untypeable, which was consistent with the clinical and autopsy findings that death was due to tampon-related toxic shock syndrome.

• Staphylococcus aureus: An old pathogen with new weapons.

  Authors: Yi-Wei Tang, Charles W Stratton

  Clinics in laboratory medicine. 03/2010; 30(1):179-208.

  Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. S aureus has been able to adapt and evolve in terms of its resistance traits and virulence factors;Staphylococcus aureus has been recognized as an important human pathogen for more than 100 years. S aureus has been able to adapt and evolve in terms of its resistance traits and virulence factors; it is among the most important causes of human infections in the twenty-first century. Rapid molecular identification in the clinical microbiology laboratory of these resistance and virulence factors expressed by S aureus will play an important role in the future in decreasing the morbidity and mortality of infections. This article addresses the emerging aspects of infections caused by S aureus, including microbiology, epidemiology, clinical presentation, pathogenesis, diagnosis, treatment and prognosis, and immunity.

• C. Diff Quik Chek Complete EIA Provides a Reliable First-Line Method for Detection of Clostridium difficile in Stool Specimens.

  Authors: Criziel D. Quinn, Susan E Sefers, Wisal Babiker, Ying He, Romina Alcabasa, Charles W Stratton, Karen C Carroll, Yi-Wei Tang

  Journal of clinical microbiology. 12/2009;

  We evaluated a single membrane device assay to simultaneously detect both Clostridium difficile glutamate dehydrogenase (GDH) and toxins A/B antigens against a standard that combines two PCR assaysWe evaluated a single membrane device assay to simultaneously detect both Clostridium difficile glutamate dehydrogenase (GDH) and toxins A/B antigens against a standard that combines two PCR assays and cytotoxigenic culture. Results with dual GDH and toxins A/B antigen positives and negatives can be reported immediately as true positives and negatives, respectively. Specimens with discrepant results between GDH and toxins A/B, which comprised 13.2% of specimens, need to be re-tested.

• Detection and Identification of Ehrlichia species in Blood Using PCR and Electrospray Ionization Mass Spectrometry.

  Authors: Mark W Eshoo, Chris D Crowder, Haijing Li, Heather E Matthews, Shufang Meng, Susan E Sefers, Ranga Sampath, Charles W Stratton, Lawrence B Blyn, David J Ecker, Yi-Wei Tang

  Journal of clinical microbiology. 12/2009;

  Rapid detection and identification of Ehrlichia species improves clinical outcome in patients suspected of ehrlichiosis. We describe an assay that employs multi-locus PCR and electrospray massRapid detection and identification of Ehrlichia species improves clinical outcome in patients suspected of ehrlichiosis. We describe an assay that employs multi-locus PCR and electrospray mass spectrometry (PCR/ESI-MS) to detect and identify Ehrlichia species directly from blood specimens. The results were compared to a colorimetric microtiter PCR (PCR-EIA) being used as a diagnostic assay. Among 213 whole blood samples collected from patients who were clinically suspected of ehrlichiosis from May 1 to August 1, 2008 at Vanderbilt University Hospital, 40 were positive for an Ehrlichia species by the PCR/ESI-MS, giving a positive rate of 18.8%. In comparison to the PCR-EIA, the PCR/ESI-MS possessed sensitivity, specificity and positive and negative predictive values of 95.0%, 98.8%, 95.0%, and 98.8%, respectively. The 38 specimens that were positive for Ehrlichia by both PCR/ESI-MS and PCR-EIA were further characterized to the species level with 100% agreement between the two assays. In addition, Rickettsia rickettsii was detected by PCR/ESI-MS from four specimens which were confirmed retrospectively by serology and PCR-EIA. In three specimens, the PCR/ESI-MS assay identified Pseudomonas aeruginosa, Neisseria meningitidis, and Staphylococcus aureus; these were confirmed by culture and/or clinical diagnosis as being clinically relevant. From specimen processing to result reporting, the PCR/ESI-MS assay can be completed within six hours, providing another laboratory tool for the diagnosis of ehrlichiosis. Moreover, this system may provide rapid detection and identification of additional pathogens directly from blood specimens.

• Identification and Differentiation of Clinically Relevant Mycobacterium Species Directly from Acid Fast Bacilli-Positive Culture Broth.

  Authors: Haijing Li, Vedat Turhan, Laxmi Chokhani, Charles W Stratton, Sherry A. Dunbar, Yi-Wei Tang

  Journal of clinical microbiology. 09/2009;

  Mycobacterium species cause a variety of clinical diseases some of which may be species-specific. Therefore, it is clinically desirable to rapidly identify and differentiate mycobacterial isolates toMycobacterium species cause a variety of clinical diseases some of which may be species-specific. Therefore, it is clinically desirable to rapidly identify and differentiate mycobacterial isolates to species level. We developed a rapid and high-throughput system, MycoID, to identify and speciate Mycobacterium species directly from acid-fast bacilli (AFB)-positive mycobacterial culture broth. The MycoID system incorporated a broad-range PCR followed by suspension-array hybridization to identify 17 clinically-relevant mycobacterial complexes, groups and species in one single reaction. We evaluated a total of 271 AFB-positive culture broth specimens, which were identified by reference standard methods in combination with biochemical and molecular tests. The overall identification agreement between the standard and the MycoID system was 89.7% (perfect match) or 97.8% (one match in co-detection). In comparison to the standard, the MycoID system possessed an overall sensitivity of 97.1% and specificity of 98.8%. The 159 M. avium-intracellulare complex (MAI) isolates were further speciated by the MycoID as M. avium (n=98, 61.1%), M. intracellulare (n=57, 35.8%) and mixed M. avium and M. intracellulare (n=2, 1.3%). M. avium was recovered more frequently from sterile sites than M. intracellulare (OR=4.6, p=0.0092). The entire MycoID procedure, including specimen processing, can be completed within five hours, providing rapid and reliable identification and differentiation of mycobacterium species that is amenable to automation. Additional differentiation of MAI into M. avium and M. intracellulare may provide a tool to better understand the role of MAI in human disease.

• Sensitive, qualitative detection of human herpesvirus-6 and simultaneous differentiation of variants A and B.

  Authors: Haijing Li, Shufang Meng, Susan M Levine, Charles W Stratton, Yi-Wei Tang

  Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 07/2009;

  BACKGROUND: The current limitations of laboratory testing for the detection of human herpesvirus virus 6 (HHV-6) in clinical specimens with low HHV-6 viral loads make this area a priority for furtherBACKGROUND: The current limitations of laboratory testing for the detection of human herpesvirus virus 6 (HHV-6) in clinical specimens with low HHV-6 viral loads make this area a priority for further research and development. OBJECTIVES: To develop and validate a sensitive qualitative assay for simultaneous HHV-6 detection and variant differentiation. METHODS: We developed a diagnostic procedure, which combines a magnetic bead-based nucleic acid extraction, PCR amplification, and colorimetric microtiter plate identification (MAG-PCR-EIA), for the sensitive detection of HHV-6 and the simultaneous differentiation of HHV-6A and HHV-6B. RESULTS: Analytic sensitivities of the MAG-PCR-EIA assay were 10 copies per reaction for both HHV-6A and HHV-6B variants, which is equivalent to 20 copies/ml when 1ml of clinical specimen was processed. A proficiency panel containing 11 blinded specimens covering HHV-6A viral loads from 0 to 100,000 copies was tested, and the MAG-PCR-EIA was able to detect the lowest concentration at one copy in 200mul. A panel of 27 urine specimens, which were collected from patients with and without chronic fatigue syndrome, was tested by the MAG-PCR-EIA. HHV-6 was detected in two (HHV-6A) patients who have chromosomally integrated HHV-6A and in one (HHV-6B) patient who was a healthy control and diagnosed as cervical cancer later on. The HHV-6 results did not correlate with results previously determined by HHV-6 antigenemia in urine. CONCLUSION: With large specimen volumes processed and an additional signal amplification incorporated, the MAG-PCR-EIA provides a sensitive and qualitative system for HHV-6 detection and simultaneous variant differentiation. Clinical relevance of the assay awaits further investigation.

• Qualitative and quantitative detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid from multiple sclerosis patients and controls.

  Authors: Yi-Wei Tang, Subramaniam Sriram, Haijing Li, Song-Yi Yao, Shufang Meng, William M Mitchell, Charles W Stratton

  PLoS ONE. 02/2009; 4(4):e5200.

  A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multipleA standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.

• PCR detection of Histoplasma capsulatum var. capsulatum in whole blood of a renal transplant patient with disseminated histoplasmosis.

  Authors: Julianne Qualtieri, Charles W Stratton, David R Head, Yi-Wei Tang

  Annals of clinical and laboratory science. 01/2009; 39(4):409-12.

  We report the identification of Histoplasma capsulatum var. capsulatum from whole blood in a renal transplant patient with disseminated histoplasmosis using colorimetric microtiter-plate PCR. ThisWe report the identification of Histoplasma capsulatum var. capsulatum from whole blood in a renal transplant patient with disseminated histoplasmosis using colorimetric microtiter-plate PCR. This modality demonstrated utility in reaching a definitive diagnosis in a timely manner. Blood fungal cultures in this case remained negative, suggesting that molecular assays may facilitate the laboratory diagnosis of disseminated histoplasmosis.

• Clonal Dissemination of Staphylococcus epidermidis on an Oncology Ward.

  Authors: Kenneth L Muldrew, Yi-Wei Tang, Haijing Li, Charles W Stratton

  Journal of clinical microbiology. 08/2008;

  Coagulase negative staphylococci (CoNS) are the main cause of catheter-related infections especially among immunosuppressed and neutropenic patients as well as a source of bacterial contamination inCoagulase negative staphylococci (CoNS) are the main cause of catheter-related infections especially among immunosuppressed and neutropenic patients as well as a source of bacterial contamination in blood cultures. Using biochemical identification and pulsed field gel electrophoresis (PFGE), we sought to identify possible clonal isolates of bacteremia in patients with central lines on an oncology ward (OW) with comparison to isolates that were recovered by venipuncture from an adult emergency room (ER). A total of 243 CoNS isolates were identified to species from the OW (126) and ER (117) with Staphylococcus epidermidis being the most common isolates (OW=79.4% and ER=45.3%). PFGE demonstrated a predominant clone of S. epidermidis (major subtype A) which was 35.5 (OR 95% CI=4.7-267.0, P<0.00001) times more likely to be present in the OW versus the ER. These (CoNS or major subtype A) isolates were more frequently resistant to gentamycin (OR=2.83; 95%CI=1.23-6.53; p=0.016) and less frequently resistant to trimethoprim/sulfamethoxazole (OR=0.38; 95%CI=0.18-0.80; P=0.013). Subset analysis of S. epidermidis isolates 2 years after the study period showed the persistence of the clone of major subtype A within the OW. This study demonstrates the presence of a predominant clone among central line isolates from an OW that is not present in CoNS venipuncture isolates from an ER.