Haijing Li

Publications of Haijing Li

• 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.

• Methicillin-resistant Staphylococcus aureus bloodstream infections in a Japanese University Hospital between 1987 and 2004.

  Authors: Tohru Takata, Motoyasu Miyazaki, Haijing Li, Mimi Healy, Stacie Frye, Koko Tanaka, Shuji Hara, Hidetoshi Kamimura, Hisae Yoshimura, Akira Matsunaga, Daiki Ohta, Hiroyasu Ishikura, Kazuo Tamura, Yi-Wei Tang

  Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy. 10/2011;

  Methicillin-resistant Staphylococcus aureus (MRSA) infections have been the most common cause of nosocomial infections in Japan, but their genetic characteristics related to bloodstream infectionsMethicillin-resistant Staphylococcus aureus (MRSA) infections have been the most common cause of nosocomial infections in Japan, but their genetic characteristics related to bloodstream infections have not been well studied. The aim of this study was to investigate a comprehensive molecular characterization of MRSA blood isolates during the historical 18-year study period between 1987 and 2004 in a tertiary care university hospital. A total of 137 MRSA isolates recovered from the blood of inpatients at Fukuoka University Hospital were analyzed. Clinical information and antimicrobial susceptibility profiles were reviewed, and staphylococcal chromosomal cassette mec (SCCmec), accessory gene regulator (agr), and a battery of bacterial genes were tested by PCR-based assays. The relatedness of these isolates was determined by the repetitive sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). Although low numbers of agr type III/SCCmec type IV isolates circulated between 1987 and 1992, agr type II/SCCmec type II isolates started circulating in 1993 and were responsible for the increased MRSA isolates until 2004. The rep-PCR and PFGE identified 104 epidemic and 33 sporadic isolates. Among the 104 epidemic isolates, six major rep-PCR/PFGE types were identified, which occupied 67.3% of epidemic isolates. The SCCmec type II and agr type II isolates were observed in significantly higher proportion in epidemic isolates than in sporadic isolates (P = 0.0318, P = 0.0123, respectively). In contrast, SCCmec type IV strains were observed in significantly higher proportion in sporadic isolates than in epidemic isolates (P = 0.0494). Although isolates with sec were detected in higher rates in epidemic isolates (P = 0.0397), seh was detected in higher rates in sporadic isolates (P = 0.0350). Multivariate logistic regression analysis with forward stepping revealed that SCCmec type II was independently associated with epidemic isolates (P = 0.0067; odds ratio, 1.75; 95% confidence interval, 1.17-2.64). These data indicated that SCCmec type II MRSA isolates were responsible for the increased MRSA bloodstream infections for inpatients during the 18-year study period in the hospital.

• 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.

• Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and database for identification of Legionella species.

  Authors: Ying He, Tsung C Chang, Haijing Li, Gongyi Shi, Yi-Wei Tang

  Canadian journal of microbiology. 07/2011; 57(7):533-8.

  More than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate betweenMore than 20 species of Legionella have been identified in relation to human infections. Rapid detection and identification of Legionella isolates is clinically useful to differentiate between infection and contamination and to determine treatment regimens. We explored the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Biotyper system (Bruker Daltonik GmbH, Bremen, Germany) for the identification of Legionella species. The MALDI MS spectra were generated and compared with the Biotyper database, which includes 25 Legionella strains covering 22 species and four Legionella pneumophila serogroups. A total of 83 blind-coded Legionella strains, consisting of 54 reference and 29 clinical strains, were analyzed in the study. Overall, the Biotyper system correctly identified 51 (61.4%) of all strains and isolates to the species level. For species included in the Biotyper database, the method identified 51 (86.4%) strains out of 59 Legionella strains to the correct species level, including 24 (100%) L. pneumophila and 27 (77.1%) non-L. pneumophila strains. The remaining 24 Legionella strains, belonging to species not covered by the Biotyper database, were either identified to the Legionella genus level or had no reliable identification. The Biotyper system produces constant and reproducible MALDI MS spectra for Legionella strains and can be used for rapid and accurate Legionella identification. More Legionella strains, especially the non-L. pneumophila strains, need to be included in the current Biotyper database to cover varieties of Legionella species and to increase identification accuracy.

• 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.

• Molecular characterization of drug-resistant Mycobacterium tuberculosis isolates circulating in China by multilocus PCR and electrospray ionization mass spectrometry.

  Authors: Feifei Wang, Christian Massire, Haijing Li, Lendell L Cummins, Feng Li, Jialin Jin, Xiaoping Fan, Sen Wang, Lingyun Shao, Shu Zhang, Shufang Meng, Jing Wu, Chanyi Lu, Lawrence B Blyn, Rangarajan Sampath, David J Ecker, Wenhong Zhang, Yi-Wei Tang

  Journal of clinical microbiology. 04/2011; 49(7):2719-21.

  We used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating inWe used multilocus PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) to determine the genotype and drug resistance profiles for 96 Mycobacterium tuberculosis isolates circulating in regions of high and low tuberculosis (TB) endemicity in China. The dominant principal genetic group (PGG) circulating in China was PGG1, and drug-resistant gene mutations were more diversified in the region of low rather than high TB endemicity.

• Simultaneous detection and differentiation of respiratory syncytial virus and other respiratory viral pathogens.

  Authors: Susan E Sefers, Haijing Li, Yi-Wei Tang

  Methods in molecular biology (Clifton, N.J.). 01/2011; 665:309-23.

  Rapid and accurate detection of respiratory syncytial virus (RSV) provides pathogen-specific diagnosis, allows implementation of appropriate infection control measures, and improves patientRapid and accurate detection of respiratory syncytial virus (RSV) provides pathogen-specific diagnosis, allows implementation of appropriate infection control measures, and improves patient management. One diagnostic challenge is that respiratory infections, which can be caused by several viral pathogens including RSV, usually present with similar signs and symptoms that are nearly indistinguishable by clinical diagnosis. We have described in the chapter a rapid, high-throughput laboratory technique that can detect a panel of common viral pathogens in one single reaction. With the combination of target-enriched multiplexing PCR amplification and Luminex suspension array identification, 12 common respiratory viruses, including RSV A and B, influenza virus A and B, parainfluenza virus 1, 2, 3, and 4, human metapneumovirus, rhinoviruses, enteroviruses, and SARS coronavirus, are detected and differentiated simultaneously within five hours.

• 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.

• New technology for rapid molecular diagnosis of bloodstream infections.

  Authors: David J Ecker, Rangarajan Sampath, Haijing Li, Christian Massire, Heather E Matthews, Donna Toleno, Thomas A Hall, Lawrence B Blyn, Mark W Eshoo, Raymond Ranken, Steven A Hofstadler, Yi-Wei Tang

  Expert review of molecular diagnostics. 05/2010; 10(4):399-415.

  Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstreamTechnologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.

• 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.

• Nasal colonization and clonal transmission of methicillin-susceptible Staphylococcus aureus in Chinese military volunteers.

  Authors: Fen Qu, Enbo Cui, Tongsheng Guo, Haijing Li, Suming Chen, Liming Liu, Wang Han, Chunmei Bao, Yuanli Mao, Yi-Wei Tang

  Journal of clinical microbiology. 11/2009;

  Military facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed theMilitary facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed the prevalence of S. aureus nasal colonization among Chinese military volunteers in two camps in the Beijing area. Antimicrobial resistance patterns, risk factors for colonization and transmission patterns using pulsed-field gel electrophoresis were also evaluated. From May to July 2007, 1,044 nasal swabs were collected from military volunteers from suburban (560) and urban (484) camps. A total of 209 S. aureus isolates were recovered of which all methicillin susceptible. Independent factors associated with methicillin-susceptible S. aureus (MSSA) nasal colonization included younger age (OR=1.51, 95% CI=1.03-2.21, P=0.0347), higher education (OR=1.38, 95% CI=1.10-1.73, P=0.0056), shorter length of service (OR=1.74, 95% CI=1.28-2.36, P=0.0004), non-smoking (OR=1.61, 95% CI=1.14-2.28, P=0.0069), and inactive participation of social events (OR=2.40, 95% CI=1.25-5.49, P=0.0082). Among 209 MSSA isolates, 126 (60.3%) were determined to be epidemic and a total of 12 genotypeswere identified of which four (90, 71.4%) represented the majority of strains. Length of service and camp location were statistically related to the four major MSSA genotype clonal transmissions. Our data indicated that MSSA, not MRSA, nasal colonization and clonal transmission occur in healthy military volunteers in Beijing. Younger, female, non-smoking, volunteers with higher education and inactive no active participation of social events of social events who served shorter are at higher risk for nasal MSSA carriage.

• 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.

• Monitoring Therapeutic Efficacy by Real-Time Detection of Mycobacterium tuberculosis mRNA in Sputum.

  Authors: Nino Mdivani, Haijing Li, Maka Akhalaia, Medea Gegia, Leila Goginashvili, Douglas S Kernodle, George Khechinashvili, Yi-Wei Tang

  Clinical chemistry. 08/2009;

  BACKGROUND: Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growthBACKGROUND: Current laboratory methods for monitoring the response to therapy for tuberculosis (TB) rely on mycobacterial culture. Their clinical usefulness is therefore limited by the slow growth rate of Mycobacterium tuberculosis. Rapid methods to reliably quantify the response to anti-TB drugs are desirable. METHODS: We developed 2 real-time PCR assays that use hydrolysis probes to target DNA of the IS6110 insertion element and mRNA for antigen 85B. The nucleic acids are extracted directly from concentrated sputum samples decontaminated with sodium hydroxide and N-acetyl-l-cysteine. We prospectively compared these assays with results obtained by sputum mycobacterial culture for patients receiving anti-TB therapy. RESULTS: Sixty-five patients with newly diagnosed TB and receiving a standardized first-line anti-TB drug regimen were evaluated at week 2 and at months 1, 2, and 4 after therapy initiation. Both the DNA PCR assay (98.5% positive) and the mRNA reverse-transcription PCR (RT-PCR) assay (95.4% positive) were better than standard Ziehl-Neelsen staining techniques (83.1%) for detecting M. tuberculosis in culture-positive sputum samples. The overall agreement between culture and mRNA RT-PCR results for all 286 sputum samples was 87.1%, and compared with culture, the mRNA RT-PCR assay's diagnostic sensitivity and specificity were 85.2% and 88.6%, respectively. For monitoring efficacy of therapy, mRNA RT-PCR results paralleled those of culture at the follow-up time points. CONCLUSIONS: The continued presence of viable M. tuberculosis according to culture and results obtained by RT-PCR analysis of antigen 85B mRNA correlated clinically with resistance to anti-TB drugs, whereas the DNA PCR assay showed a high false-positive rate. This mRNA RT-PCR assay may allow rapid monitoring of the response to anti-TB therapy.

• 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.

• Detection of viruses in human adenoid tissues using multiplex-PCR.

  Authors: Masatoki Sato, Haijing Li, Mine R Ikizler, Jay A. Werkhaven, John V Williams, James D Chappell, Yi-Wei Tang, Peter F Wright

  Journal of clinical microbiology. 01/2009;

  We detected a high frequency of viruses by polymerase chain reaction (PCR) in adenoids obtained from children without acute respiratory symptoms. Our results suggest that persistent/latent viralWe detected a high frequency of viruses by polymerase chain reaction (PCR) in adenoids obtained from children without acute respiratory symptoms. Our results suggest that persistent/latent viral infection in the respiratory tract confounds interpretation of the association of PCR pathogen detection in these sources with acute respiratory infection.

• 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.