Niaz Banaei

All Publications

• Clinical Impact of Metagenomic Next-Generation Sequencing of Plasma Cell-Free DNA for the Diagnosis of Infectious Diseases: A Multicenter Retrospective Cohort Study. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Hogan, C. A., Yang, S., Garner, O. B., Green, D. A., Gomez, C. A., Dien Bard, J., Pinsky, B. A., Banaei, N. 2020

  Abstract

  Metagenomic next-generation sequencing (mNGS) of plasma cell-free DNA has emerged as an attractive diagnostic modality allowing broad-range pathogen detection, noninvasive sampling, and earlier diagnosis. However, little is known about its real-world clinical impact as used in routine practice.We performed a retrospective cohort study of all patients for whom plasma mNGS (Karius test) was performed for all indications at 5 U.S. institutions over 1.5 years. Comprehensive chart review was performed, and standardized assessment of clinical impact of the mNGS based on the treating team's interpretation of Karius results and patient management was established.A total of 82 Karius tests were evaluated, from 39 (47.6%) adults and 43 (52.4%) children and a total of 53 (64.6%) immunocompromised patients. Karius positivity rate was 50/82 (61.0%), with 25 (50.0%) showing two or more organisms (range, 2-8). The Karius test results led to positive impact in 6 (7.3%), negative impact in 3 (3.7%), no impact in 71 (86.6%), and was indeterminate in 2 (2.4%). Cases with positive Karius result and clinical impact involved bacteria and/or fungi but not DNA viruses or parasites. In 10 patients who underwent 16 additional repeated tests, only one was associated with clinical impact.The real-world impact of the Karius test as currently used in routine clinical practice is limited. Further studies are needed to identify high-yield patient populations, define the complementary role of mNGS to conventional microbiological methods and how best to integrate mNGS into current testing algorithms.

  View details for DOI 10.1093/cid/ciaa035

  View details for PubMedID 31942944

• Simple Processing of Formalin-Fixed Paraffin-Embedded Tissue for Accurate Testing with the Xpert MTB/RIF assay. Journal of clinical microbiology Budvytiene, I., Banaei, N. 2019

  Abstract

  Extrapulmonary tuberculosis (TB) is a common presentation of TB, which accounted for 15% of new TB cases in 2018 (1)..

  View details for DOI 10.1128/JCM.01905-19

  View details for PubMedID 31852763

• Intestinal microbiota domination under extreme selective pressures characterized by metagenomic read cloud sequencing and assembly. BMC bioinformatics Kang, J. B., Siranosian, B. A., Moss, E. L., Banaei, N., Andermann, T. M., Bhatt, A. S. 2019; 20 (Suppl 16): 585

  Abstract

  BACKGROUND: Low diversity of the gut microbiome, often progressing to the point of intestinal domination by a single species, has been linked to poor outcomes in patients undergoing hematopoietic cell transplantation (HCT). Our ability to understand how certain organisms attain intestinal domination over others has been restricted in part by current metagenomic sequencing technologies that are typically unable to reconstruct complete genomes for individual organisms present within a sequenced microbial community. We recently developed a metagenomic read cloud sequencing and assembly approach that generates improved draft genomes for individual organisms compared to conventional short-read sequencing and assembly methods. Herein, we applied metagenomic read cloud sequencing to four stool samples collected longitudinally from an HCT patient preceding treatment and over the course of heavy antibiotic exposure.RESULTS: Characterization of microbiome composition by taxonomic classification of reads reveals that that upon antibiotic exposure, the subject's gut microbiome experienced a marked decrease in diversity and became dominated by Escherichia coli. While diversity is restored at the final time point, this occurs without recovery of the original species and strain-level composition. Draft genomes for individual organisms within each sample were generated using both read cloud and conventional assembly. Read clouds were found to improve the completeness and contiguity of genome assemblies compared to conventional assembly. Moreover, read clouds enabled the placement of antibiotic resistance genes present in multiple copies both within a single draft genome and across multiple organisms. The occurrence of resistance genes associates with the timing of antibiotics administered to the patient, and comparative genomic analysis of the various intestinal E. coli strains across time points as well as the bloodstream isolate showed that the subject's E. coli bloodstream infection likely originated from the intestine. The E. coli genome from the initial pre-transplant stool sample harbors 46 known antimicrobial resistance genes, while all other species from the pre-transplant sample each contain at most 5 genes, consistent with a model of heavy antibiotic exposure resulting in selective outgrowth of the highly antibiotic-resistant E. coli.CONCLUSION: This study demonstrates the application and utility of metagenomic read cloud sequencing and assembly to study the underlying strain-level genomic factors influencing gut microbiome dynamics under extreme selective pressures in the clinical context of HCT.

  View details for DOI 10.1186/s12859-019-3073-1

  View details for PubMedID 31787070

• Reproducibility of positive results for rare pathogens on the FilmArray GI Panel DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Hitchcock, M. M., Hogan, C. A., Budvytiene, I., Banaei, N. 2019; 95 (1): 10?14

  View details for DOI 10.1016/j.diagmicrobio.2019.03.013

  View details for Web of Science ID 000483427000003

• Eremothecium coryli bloodstream infection in a patient with acute myeloid leukemia: first case report of human infection DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Multani, A., Rustagi, A., Epstein, D. J., Gomez, C. A., Budvytiene, I., Banaei, N., Brown, J. M., Liu, A. Y. 2019; 95 (1): 77?79

  View details for DOI 10.1016/j.diagmicrobio.2019.03.011

  View details for Web of Science ID 000483427000016

• Comparing QuantiFERON-TB Gold Plus with other tests to diagnose Mycobacterium tuberculosis infection. Journal of clinical microbiology Venkatappa, T. K., Punnoose, R., Katz, D. J., Higgins, M. P., Banaei, N., Graviss, E. A., Belknap, R. W., Ho, C. S., Tuberculosis Epidemiologic Studies Consortium 2019

  Abstract

  The fourth generation QuantiFERON test for tuberculosis infection, QuantiFERON-TB Gold Plus (QFT-Plus) has replaced the earlier version, QuantiFERON-TB Gold In-Tube (QFT-GIT). A clinical need exists for information about agreement between QFT-Plus and other tests.We conducted this study to assess agreement of test results for QFT-Plus with those of QuantiFERON-TB Gold In-Tube (QFT-GIT), T-SPOT.TB (T-SPOT) and the tuberculin skin test (TST).Persons at high risk of LTBI and/or progression to TB disease were enrolled at the 10 sites of the Tuberculosis Epidemiologic Studies Consortium from October 2016 through May 2017; each participant received all four tests. Cohen's kappa (kappa) and Wilcoxon signed rank test compared qualitative and quantitative results of QFT-Plus with the other tests.Test results for 506 participants showed 94% agreement between QFT-Plus and QFT-GIT, with 19% positive and 75% negative results. When the tests disagreed, it was most often in the direction of QFT-GIT negative/QFT-Plus positive. QFT-Plus had similar concordance as QFT-GIT with TST (77% and 77%) and T-SPOT (92% and 91%), respectively.Conclusions: The study showed high agreement between QFT-GIT and QFT-Plus in a direct comparison. Both tests had similar agreement with TST and T-SPOT.

  View details for DOI 10.1128/JCM.00985-19

  View details for PubMedID 31462550

• Rapid antimicrobial susceptibility testing by VITEK (R) 2 directly from blood cultures in patients with Gram-negative rod bacteremia DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Hogan, C. A., Watz, N., Budvytiene, I., Banaei, N. 2019; 94 (2): 116?21

  View details for DOI 10.1016/j.diagmicrobio.2019.01.001

  View details for Web of Science ID 000469164300003

• H-1, C-13 and N-15 resonance assignments and structure prediction of translation initiation factor 1 from Clostridium difficile BIOMOLECULAR NMR ASSIGNMENTS Aguilar, F., Banaei, N., Zhang, Y. 2019; 13 (1): 91?95

  View details for DOI 10.1007/s12104-018-9858-8

  View details for Web of Science ID 000463649500019

• Rapid antimicrobial susceptibility testing by VITEK2 directly from blood cultures in patients with Gram-negative rod bacteremia. Diagnostic microbiology and infectious disease Hogan, C. A., Watz, N., Budvytiene, I., Banaei, N. 2019

  Abstract

  OBJECTIVE: Optimizing therapy for bacteremia is currently limited by the 1-2-day turnaround time required for antimicrobial susceptibility testing (AST). Here, we assess a rapid AST method with VITEK2 (bioMerieux, France) directly from positive blood cultures.METHODS: Patient-derived positive blood cultures with Gram-negative rods identified as Enterobacteriaceae and Pseudomonas aeruginosa were prospectively tested, and other blood culture bottles were spiked with carbapenem-resistant Enterobacteriaceae (CRE). Positive cultures were subjected to red blood cell lysis and centrifugation, and setup on VITEK2.RESULTS: A total of 109 patient blood cultures and 52 spiked blood cultures were tested. Overall, essential agreement was 97.7% [95% confidence interval (CI) 96.4-99.0], and categorical agreement was 96.8% (95% CI 95.0-98.6). Mean turnaround time from setup to susceptibility results for Enterobacteriaceae in the clinical cultures was 9.0 (±1.3) h.CONCLUSIONS: Direct susceptibility testing of blood cultures by VITEK2 for Enterobacteriaceae is an accurate, practical, and inexpensive diagnostic strategy for rapid automated AST.

  View details for PubMedID 30711413

• Higher Positivity Rate with Fourth-Generation QuantiFERON-TB Gold Plus Assay in Low-Risk US Health Care Workers JOURNAL OF CLINICAL MICROBIOLOGY Hogan, C. A., Tien, S., Pai, M., Banaei, N. 2019; 57 (1)

  View details for DOI 10.1128/JCM.01688-18

  View details for Web of Science ID 000455018000023

• Eremothecium coryli bloodstream infection in a patient with acute myeloid leukemia: first case report of human infection. Diagnostic microbiology and infectious disease Multani, A., Rustagi, A., Epstein, D. J., Gomez, C. A., Budvytiene, I., Banaei, N., Brown, J. M., Liu, A. Y. 2019

  Abstract

  Eremothecium coryli is a dimorphic fungus of the Saccharomycetes class. While species within this class are known to cause human infection, Eremothecium species have previously only been known as phytopathogens and never been isolated from a human sample. Here, we report the first known case of human E. coryli infection.

  View details for PubMedID 31005402

• Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. Journal of clinical microbiology Murugesan, K., Hogan, C. A., Palmer, Z., Reeve, B., Theron, G., Andama, A., Somoskovi, A., Steadman, A., Madan, D., Andrews, J., Croda, J., Sahoo, M. K., Cattamanchi, A., Pinsky, B. A., Banaei, N. 2019

  Abstract

  Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood.Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus and EBV, and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. PCR cycle threshold (CT) was used to measure amplifiable cfDNA.In spiked samples, median CT for M. tuberculosis, S. enterica, and EBV cfDNA was significantly lower in blood collected in K2EDTA than Streck and PAXgene blood collection tubes, and significantly lower in EDTA-urine than Streck-urine. Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT compared with Streck blood collection tube and urine preservative. Processing delay increased median pathogen CT for Streck and PAXgene but not K2EDTA blood samples, and for urine preserved with Streck reagent but not EDTA. Double spin compared with single spin plasma separation increased median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant, and between fresh and thawed plasma and urine after 24 weeks at -80 °C. Larger plasma and urine volume in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest large volume single spin K2EDTA-plasma and EDTA-whole urine with up to 24-hour processing delay may optimize pcfDNA detection.

  View details for DOI 10.1128/JCM.00782-19

  View details for PubMedID 31511335

• Dual reporting of Clostridioides difficile PCR and predicted toxin result based on PCR cycle threshold reduces treatment of toxin-negative patients without increases in adverse outcomes. Journal of clinical microbiology Hitchcock, M. M., Holubar, M., Hogan, C. A., Tompkins, L. S., Banaei, N. 2019

  Abstract

  Nucleic acid amplification tests are commonly used to diagnose C. difficile infection (CDI). Two-step testing with toxin enzyme immunoassay is recommended to discriminate between infection and colonization but requires additional resources. Prior studies showed that PCR cycle threshold (CT) can predict toxin positivity with high negative predictive value. Starting Oct. 2016, predicted toxin result (CT-Toxin) based on a validated cut-off was routinely reported at our facility. To evaluate the clinical efficacy of this reporting, all adult patients with positive GeneXpert PCR from Oct. 2016 through Oct. 2017 underwent chart review to measure recurrence of or conversion to a CT-Toxin+ result and 30-day all-cause mortality. There were 482 positive PCR tests in 430 unique patients, 282 CT-Toxin+ and 200 CT-Toxin-. Patient characteristics were similar at testing, though CT-Toxin+ patients had a higher WBC count (12.5 v. 9.3 k/?L; p=0.001). All cases (n=21) of fulminant CDI had a CT-Toxin+ result. Index CT-Toxin+ patients were significantly more likely to have a CT-Toxin+ result within 90 days than CT-Toxin- patients (17.4% [n=49] v. 8.0% [n=16]; p=0.003). Thirty-day all-cause mortality was higher in CT-Toxin- patients (11.1% v. 6.8%; p=0.1), though no deaths in CT-Toxin- patients were directly attributable to CDI. Of 200 CT-Toxin- patients, 51.5% (n=103) were treated for CDI. The rate of conversion to a CT-Toxin+ result (8.8% v. 7.2%; p=0.8) and all-cause mortality (8.8% v. 13.4%; p=0.3) were similar between treated and untreated CT-Toxin- patients. CT-based toxin prediction may identify patients at higher risk for CDI-related complications and reduce treatment among CT-Toxin- patients.

  View details for DOI 10.1128/JCM.01288-19

  View details for PubMedID 31511334

• Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning. Nature communications Ho, C. S., Jean, N., Hogan, C. A., Blackmon, L., Jeffrey, S. S., Holodniy, M., Banaei, N., Saleh, A. A., Ermon, S., Dionne, J. 2019; 10 (1): 4927

  Abstract

  Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.

  View details for DOI 10.1038/s41467-019-12898-9

  View details for PubMedID 31666527

• Reproducibility of positive results for rare pathogens on the FilmArray GI Panel. Diagnostic microbiology and infectious disease Hitchcock, M. M., Hogan, C. A., Budvytiene, I., Banaei, N. 2019

  Abstract

  Though the FilmArray GI Panel has a reported aggregate specificity and reproducibility of >97% and?>?99%, respectively, the reproducibility is less understood in clinical practice. We measured the reproducibility of positive results for low-prevalence pathogens. Samples with positive results for selected targets were repeated using a different FilmArray module. Overall, 331 of 373 (89%) results were reproducible. Giardia lamblia (57/57, 100%), Cryptosporidium spp. (61/63, 97%), Cyclospora cayetanensis (34/35, 97%), Plesiomonas shigelloides (17/18, 94%), and Rotavirus A (76/77, 99%) were highly reproducible, while Adenovirus F40/41 (38/54, 70%), Vibrio spp. (8/10, 80%), V. cholerae (3/8, 37.5%), and Yersinia enterocolitica (36/50, 72%) were poorly reproducible. Review of 38 patients with nonreproducible results showed that 19 (50%) had evidence of gastroenteritis and only 6 (16%) had possible infection with the organism that showed a nonreproducible result. Higher false-positive rates with certain targets on FAGP emphasize the need for diagnostic stewardship.

  View details for PubMedID 31029490

• Diverse Bloodstream Pathogens Are Identified in the Gut Microbiome Prior to Infection in Hematopoietic Stem Cell Transplantation Recipients Andermann, T. M., Tamburini, F., Tkachenko, E., Senchyna, F., Banaei, N., Bhatt, A. S. AMER SOC HEMATOLOGY. 2018

  View details for DOI 10.1182/blood-2018-99-113089

  View details for Web of Science ID 000454837606080

• Clinical Impact of Clostridium difficile PCR Cycle Threshold-Predicted Toxin Reporting in Pediatric Patients. Journal of the Pediatric Infectious Diseases Society Schwenk, H. T., Bio, L. L., Kruger, J. F., Banaei, N. 2018

  Abstract

  Background: Reliance on tests that detect only the presence of toxigenic Clostridium difficile can result in overdiagnosis and overtreatment of C difficile infection (CDI). The C difficile polymerase chain reaction (PCR) cycle threshold (CT) can sensitively predict the presence of free C difficile toxins; however, the clinical application for this testing strategy remains unexplored. We evaluated the impact of dual PCR and toxin result reporting, as predicted by the CT, on CDI management and outcomes in children.Methods: Before the intervention, results for C difficile testing at Lucile Packard Children's Hospital Stanford were reported as PCR positive (PCR+) or negative (PCR-) according to the GeneXpert C diff Epi tcdB PCR assay (Cepheid, Sunnyvale, California). Beginning October 5, 2016, the presence of free toxins, as predicted by the CT, was reported also. The CDI treatment rates 1 year before and 18 months after implementation of toxin reporting were compared. Demographic and treatment-related data were collected, and patient outcomes were followed up 8 weeks later.Results: CDI treatment decreased 22% after the intervention (96% [preintervention] vs 74% [postintervention]; P < .001). During the postintervention period, there were 152 PCR+C difficile results, and 94 (62%) of them were toxin positive (toxin+) according to the CT. Of the 58 PCR+/toxin-negative (toxin-) results, 38 (66%) did not result in CDI treatment. Seven (18%) of the untreated PCR+/toxin- patients underwent repeat testing within 8 weeks, and 5 (13%) of them were subsequently PCR+/toxin+ and treated. No CDI-related complications were identified.Conclusions: Addition of the CT-predicted C difficile toxin result to PCR reporting reduces the proportion of PCR+ children treated for CDI.

  View details for PubMedID 30476169

• Towards the development of a cfDNA-based in-vitro diagnostic test for infectious diseases: A review of evidence for tuberculosis. Journal of clinical microbiology Fernandez-Carballo, B. L., Broger, T., Wyss, R., Banaei, N., Denkinger, C. M. 2018

  Abstract

  Detection of circulating free DNA (cfDNA) has transformed the field of oncology and prenatal diagnostics. Clinical application of cfDNA for disease diagnosis and monitoring, however, is relatively recent in the field of infectious disease. The potential of cfDNA as a non-invasive diagnostic and monitoring tool is especially promising for tuberculosis (TB) as it enables detection of both pulmonary and extrapulmonary TB from easily accessible urine and/or blood samples from any age group. However, despite the potential of cfDNA detection to identify TB, very few studies are described in the literature to date. A comprehensive search of the literature identified 15 studies that report detecting M. tuberculosis DNA in the blood and urine of TB patients with non-genitourinary disease, but in only six of them were the methodological steps considered suitable for cfDNA isolation and detection. The sensitivities and specificities for diagnosis of pulmonary and extrapulmonary TB cases reported in these six studies are highly variable, falling in the range of 29-79% and 67-100%, respectively. While most studies could not meet the performance requirements of the high-priority target product profiles (TPP) published by the World Health Organization (WHO), the study results nonetheless show promise for a point-of-care detection assay. Better designed prospective studies, using appropriate samples, will be required to validate cfDNA as a TB biomarker.

  View details for DOI 10.1128/JCM.01234-18

  View details for PubMedID 30404942

• Liquid biopsy for infectious diseases: sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Hong, D. K., Blauwkamp, T. A., Kertesz, M., Bercovici, S., Truong, C., Banaei, N. 2018; 92 (3): 210?13

  Abstract

  Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infected tissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The noninvasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.

  View details for PubMedID 30017314

• Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology JOURNAL OF CLINICAL MICROBIOLOGY Sandlund, J., Bartolome, A., Almazan, A., Tam, S., Biscocho, S., Abusali, S., Bishop, J. J., Nolan, N., Estis, J., Todd, J., Young, S., Senchyna, F., Banaei, N. 2018; 56 (11)

  Abstract

  Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

  View details for PubMedID 30158195

• 1H, 13C and 15N resonance assignments and structure prediction of translation initiation factor 1 from Clostridium difficile. Biomolecular NMR assignments Aguilar, F., Banaei, N., Zhang, Y. 2018

  Abstract

  Clostridium difficile is a gram-positive, toxin-producing, anaerobic bacterium whose virulence factors and mechanisms of pathogenesis require further investigation. C. difficile infections (CDI) result in the severe and potentially fatal gastrointestinal diseases pseudomembranous colitis and toxic megacolon following extensive broad spectrum antibiotic treatment. The increasing C. difficile fatalities are a result of the bacteria's growing antibiotic resistance and consequential CDI recurrence, which led to the unmet need for new CDI treatment. Bacterial protein synthesis is an essential metabolic process and an effective target for antibacterial agents. Translation initiation factor 1 from C. difficile (Cd-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis. Here we report the complete NMR 1H, 13C and 15N chemical shift assignments of Cd-IF1 as the basis for NMR structure determination and interaction studies. Secondary structure analyses have identified five beta-strands and one short alpha-helix arranged in the sequential order beta1-beta2-beta3-alpha1-beta4-beta5, which is supported by 15N-{1H} heteroNOEs. The assigned chemical shifts were used to conduct structure prediction by CS-Rosetta. The predicted structure suggests that Cd-IF1 adopts the typical beta-barrel structure and is composed of an oligomer-binding motif.

  View details for PubMedID 30370502

• Higher positivity rate with 4th generation QuantiFERON-TB Gold Plus assay in low-risk U.S. healthcare workers. Journal of clinical microbiology Hogan, C., Tien, S., Pai, M., Banaei, N. 2018

  Abstract

  We report a significant increase in positivity rates among low-risk health care workers (HCW) undergoing annual TB testing at our health system after the 4th generation QuantiFERON-TB Gold Plus (QFT-Plus) assay replaced QuantiFERON-TB Gold (QFT)..

  View details for PubMedID 30355762

• Precision identification of diverse bloodstream pathogens in the gut microbiome. Nature medicine Tamburini, F. B., Andermann, T. M., Tkachenko, E., Senchyna, F., Banaei, N., Bhatt, A. S. 2018

  Abstract

  A comprehensive evaluation of every patient with a bloodstream infection includes an attempt to identify the infectious source. Pathogens can originate from various places, such as the gut microbiota, skin and the external environment. Identifying the definitive origin of an infection would enable precise interventions focused on management of the source1,2. Unfortunately, hospital infection control practices are often informed by assumptions about the source of various specific pathogens; if these assumptions are incorrect, they lead to interventions that do not decrease pathogen exposure3. Here, we develop and apply a streamlined bioinformatic tool, named StrainSifter, to match bloodstream pathogens precisely to a candidate source. We then leverage this approach to interrogate the gut microbiota as a potential reservoir of bloodstream pathogens in a cohort of hematopoietic cell transplantation recipients. We find that patients with Escherichia coli and Klebsiella pneumoniae bloodstream infections have concomitant gut colonization with these organisms, suggesting that the gut may be a source of these infections. We also find cases where typically nonenteric pathogens, such as Pseudomonas aeruginosa and Staphylococcus epidermidis, are found in the gut microbiota, thereby challenging the existing informal dogma of these infections originating from environmental or skin sources. Thus, we present an approach to distinguish the source of various bloodstream infections, which may facilitate more accurate tracking and prevention of hospital-acquired infections.

  View details for PubMedID 30323331

• Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016. Diagnostic microbiology and infectious disease Senchyna, F., Gaur, R. L., Sandlund, J., Truong, C., Tremintin, G., Kultz, D., Gomez, C. A., Tamburini, F. B., Andermann, T., Bhatt, A., Tickler, I., Watz, N., Budvytiene, I., Shi, G., Tenover, F. C., Banaei, N. 2018

  Abstract

  The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam; blaOXA-48 like-positive isolates were 100% susceptible to ceftazidime-avibactam; and metallo beta-lactamase-positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, and ceftolozane-tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer beta-lactamase inhibitors.

  View details for PubMedID 30482638

• Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe SCIENCE TRANSLATIONAL MEDICINE Cheng, Y., Xie, J., Lee, K., Gaur, R. L., Song, A., Dai, T., Ren, H., Wu, J., Sun, Z., Banaei, N., Akin, D., Rao, J. 2018; 10 (454)

  Abstract

  Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the ?-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-?-d-ribose 2'-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients' sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.

  View details for PubMedID 30111644

• Spiking of intravenous bags does not cause time-dependent microbial contamination: a preliminary report. Infection control and hospital epidemiology Brock-Utne, J. G., Smith, S. C., Banaei, N., Chang, S., Alejandro-Harper, D., Jaffe, R. A. 2018: 1?2

  View details for PubMedID 29961441

• Development of colorimetric sensor array for diagnosis of tuberculosis through detection of urinary volatile organic compounds. Diagnostic microbiology and infectious disease Sandlund, J., Lim, S., Queralto, N., Huang, R., Yun, J., Taba, B., Song, R., Odero, R., Ouma, G., Sitati, R., Murithi, W., Cain, K. P., Banaei, N. 2018

  Abstract

  BACKGROUND: Top priorities for tuberculosis control and elimination include a simple, low-cost screening test using sputum and a non-sputum-based test in patients that do not produce sputum. The aim of this study was to evaluate the performance of a colorimetric sensor array (CSA) test, for analysis of volatile organic compounds in urine, in the diagnosis of pulmonary TB.MATERIAL AND METHODS: Urine samples were collected from individuals suspected of having pulmonary TB in Western Kenya. Reference methods included MGIT culture and/or Xpert MTB/RIF nucleic acid amplification test on sputa. Fresh urine samples were tested with the CSA, with acid and base and without an additive. The CSA were digitally imaged, and the resulting colorimetric response patterns were used for chemometric analysis. Sensitivity, specificity, and negative (NPV) and positive predictive (PPV) values were determined for HIV-positive and HIV-negative patients.RESULTS: In HIV-negative patients, the highest accuracy was obtained in urine samples pre-treated with a base, yielding a sensitivity, specificity, PPV, and NPV of 78.3% (65/83), 69.2% (54/78), 73.0% (n/89) and 75.0% (n/72). The highest sensitivity of 79.5% was achieved using sensor data from all three test conditions at a specificity of 65.4%. In HIV-positive subjects, the sensor performance was substantially lower with sensitivity, specificity, PPV, and NPV ranging from 48.3% to 62.3%, 54.1% to 74.0%, 55.9% to 64.2%, and 60.6% to 64.9%, respectively.CONCLUSION: The CSA fingerprint of urine headspace volatiles showed moderate accuracy in diagnosing TB in HIV-negative patients, but the sensor performance dropped substantially in HIV-coinfected patients. Further development of TB-responsive CSA indicators may improve the accuracy of CSA urine assay.

  View details for PubMedID 30025968

• Evaluation of the Xpert MTB/RIF Performance on Tissues: Potential Impact on Airborne Infection Isolation at a Tertiary Cancer Care Center INFECTION CONTROL AND HOSPITAL EPIDEMIOLOGY McMillen, T., Usiak, S. C., Chen, L., Gomez, L., Ntiamoah, P., Hameed, M. R., Budvytiene, I., Banaei, N., Kamboj, M., Babady, N. 2018; 39 (4): 462?66

  Abstract

  OBJECTIVES In this study, we sought to evaluate the performance of the Xpert MTB/RIF (Cepheid) assay for the detection of Mycobacterium tuberculosis (MTB) complex DNA on fresh and formalin-fixed, paraffin-embedded (FFPE) tissue specimens from oncology patients in an area with a low prevalence of tuberculosis. We also aimed to retrospectively assess the potential impact of Xpert MTB/RIF on the duration of airborne infection isolation (AII). SETTING A 473-bed, tertiary-care cancer center in New York City. DESIGN A total of 203 tissue samples (101 FFPE and 102 fresh) were tested using Xpert MTB/RIF, including 133 pulmonary tissue samples (65.5%) and 70 extrapulmonary tissue samples (34.5%). Acid-fast bacilli (AFB) culture was used as the diagnostic gold standard. The limit of detection (LOD) and reproducibility were also evaluated for both samples types using contrived specimens. The potential impact of the Xpert MTB PCR assay on tissue samples from AII patients on AII duration was retrospectively assessed. RESULTS Using the Xpert MTB/RIF for fresh tissue specimens, the sensitivity was 50% (95% CI, 1.3%-98.7%) and the specificity was 99% (95% CI, 94.5%-99.9%). For FFPE tissue specimens, the sensitivity was 100% (95% CI, 63.1%-100%) and the specificity was 98.3% (95% CI, 95.5%-100%. The LOD was 103 colony-forming units (CFU)/mL for both fresh and FFPE tissue specimens, and the Xpert MTB/RIF was 100% reproducible at concentrations 10 times that of the LOD. With an expected turnaround time of 24 hours, the Xpert MTB PCR could decrease the duration of AII from a median of 8 days to a median of 1 day. CONCLUSIONS The Xpert MTB/RIF assay offers a valid option for ruling out Mycobacterium tuberculosis complex (MTBC) on tissue samples from oncology patients and for minimizing AII resource utilization. Infect Control Hosp Epidemiol 2018;39:462-466.

  View details for PubMedID 29444723

• Answer to the letter to the editor of MN Capoor et al. concerning "Ribosomal PCR assay of excised intervertebral discs from patients undergoing single-level primary lumbar microdiscectomy'' by Alamin TF, Munoz M, Zagel A, et al.: Eur Spine J; 2017 EUROPEAN SPINE JOURNAL Alamin, T., Munoz, M., Zagel, A., Budvytiene, I., Banaei, N. 2018; 27 (2): 518?19

  View details for PubMedID 29275521

• Trypanosoma cruzi Reactivation in the Brain. The New England journal of medicine Gomez, C. A., Banaei, N. 2018; 378 (19): 1824

  View details for DOI 10.1056/NEJMicm1703763

  View details for PubMedID 29742366

• Low Yield of FilmArray GI Panel in Hospitalized Patients with Diarrhea: an Opportunity for Diagnostic Stewardship Intervention. Journal of clinical microbiology Hitchcock, M. M., Gomez, C. A., Banaei, N. 2018; 56 (3)

  Abstract

  The FilmArray GI panel (BioFire Diagnostics, Salt Lake City, UT) is a multiplex, on-demand, sample-to-answer, real-time PCR assay for the syndromic diagnosis of infectious gastroenteritis that has become widely adopted and, in some instances, has replaced conventional stool culture and parasite exams. Conventional testing has historically been restricted among hospitalized patients due to low diagnostic yield, but it is not known whether use of the FilmArray GI panel should be circumscribed. Cary-Blair stool samples submitted for FilmArray GI panel in adult patients admitted to an academic hospital from August 2015 to January 2017 were included in this study. Of 481 tests performed >72 h after admission, 29 (6.0%) were positive, all for a single target, excludingClostridium difficileWhen follow-up tests beyond the first positive per hospitalization were excluded, 20 (4.8%) of 414 tests were positive. There was no difference in yield by immune status. Most targets detected were viral (79% of all positives [n= 23] and 70% in unique patients [n= 14]). All four cases positive for a bacterial target could not be confirmed and presentation was atypical, suggesting possible false positives. After removing potential false positives and chronic viral shedders, the yield was 3.0% (12/406). Repeat testing performed >72 h after admission and following a negative result within the first 72 h was done in 19 patients and 100% (22/22) remained negative. The FilmArray GI panel has low yield in adult patients hospitalized for >72 h, similar to conventional stool microbiology tests, and it is reasonable to restrict its use in this population.

  View details for PubMedID 29237784

• Microfluidics for Combating Antimicrobial Resistance TRENDS IN BIOTECHNOLOGY Liu, Z., Banaei, N., Ren, K. 2017; 35 (12): 1129?39

  Abstract

  The ever-growing threat of antimicrobial resistance (AMR) demands immediate countermeasures. With its novelty and enabling features including downscaled analysis, precisely controlled local environment, and enhanced speed, accuracy, and cost-efficiency, microfluidics has demonstrated potential in several key areas, including furthering our understanding of bacteria, developing better susceptibility testing tools, and overcoming obstacles in discovery and research of new antibiotics. While ample research results in the field of microfluidics are available, their transformation into practical application is still lagging far behind. We believe that the challenge of AMR will give microfluidics a much-needed opportunity to leap from research papers to true productivity, and gain wider acceptance as a mature technology.

  View details for PubMedID 29153761

• Clostridium difficile PCR Cycle Threshold Predicts Free Toxin JOURNAL OF CLINICAL MICROBIOLOGY Senchyna, F., Gaur, R. L., Gombar, S., Truong, C. Y., Schroeder, L. F., Banaei, N. 2017; 55 (9): 2651?60

  Abstract

  There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenic C. difficile in stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.

  View details for PubMedID 28615471

  View details for PubMedCentralID PMC5648702

• Detecting New Mycobacterium tuberculosis Infection Time for a More Nuanced Interpretation of QuantiFERON Conversions AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Banaei, N., Pai, M. 2017; 196 (5): 546?47

  View details for PubMedID 28763239

• Evaluation of QuantiFERON-TB Gold-Plus in Health Care Workers in a Low-Incidence Setting. Journal of clinical microbiology Moon, H., Gaur, R. L., Tien, S. S., Spangler, M., Pai, M., Banaei, N. 2017; 55 (6): 1650-1657

  Abstract

  Although launched in 2015, little is known about the accuracy of QuantiFERON-TB Gold-Plus (QFT-Plus) for diagnosis of latent M. tuberculosis infection (LTBI). Unlike its predecessor, QFT-Plus utilizes two antigen tubes to elicit an immune response from CD4(+) and CD8(+) T lymphocytes. We conducted a cross-sectional study in low-risk health care workers (HCWs) at a single U.S. center to compare QFT-Plus to QuantiFERON-TB Gold in-tube (QFT). A total of 989 HCWs were tested with both QFT and QFT-Plus. Risk factors for LTBI were obtained from a questionnaire. QFT-Plus was considered positive if either antigen tube 1 (TB1) or TB2 tested positive, per the manufacturer's recommendations, or if both TB1 and TB2 tested positive, using a conservative definition. Results were compared using Cohen's kappa and linear regression, respectively. Agreement of QFT with QFT-Plus was high, at 95.6% (95% confidence interval [CI], 94.3 to 96.9; kappa, 0.57). The majority of discordant results between QFT and QFT-Plus TB1 (84.8%) and QFT and QFT-Plus TB2 (88.6%) fell within the range of 0.2 to 0.7 IU/ml. The positivity rate in 626 HCWs with no identifiable risk factors and no self-reported history of positive LTBI tests was 2.1% (CI, 1.0 to 3.2) and 3.0% (CI, 1.7 to 4.3) with QFT and QFT-Plus, respectively. A conservative definition of a QFT-Plus-positive result yielded a positivity rate of 1.0% (CI, 0.2 to 1.7; P value of 0.0002 versus QFT-Plus and 0.07 versus QFT). On follow-up testing, of 11 HCWs with discordant QFT-Plus results, 90.9% (10/11) had a negative QFT result. The QFT-Plus assay showed a high degree of agreement with QFT in U.S. HCWs. A conservative interpretation of QFT-Plus eliminated nearly all nonreproducible positive results in low-risk HCWs. Larger studies are needed to validate the latter finding and to more clearly define conditions under which a conservative interpretation can be used to minimize nonreproducible positive results in low-risk populations.

  View details for DOI 10.1128/JCM.02498-16

  View details for PubMedID 28298455

• Malaria and Chikungunya Detected Using Molecular Diagnostics Among Febrile Kenyan Children OPEN FORUM INFECTIOUS DISEASES Waggoner, J., Brichard, J., Mutuku, F., Ndenga, B., Heath, C., Mohamed-Hadley, A., Sahoo, M. K., Vulule, J., Lefterova, M., BanaeiA, N., Mukoko, D., Pinsky, B. A., LaBeaud, A. 2017; 4 (3): ofx110

  Abstract

  In sub-Saharan Africa, malaria is frequently overdiagnosed as the cause of an undifferentiated febrile illness, whereas arboviral illnesses are presumed to be underdiagnosed.Sera from 385 febrile Kenyan children, who presented to 1 of 4 clinical sites, were tested using microscopy and real-time molecular assays for dengue virus (DENV), chikungunya virus (CHIKV), malaria, and Leptospira.Malaria was the primary clinical diagnosis for 254 patients, and an arboviral infection (DENV or CHIKV) was the primary diagnosis for 93 patients. In total, 158 patients (41.0%) had malaria and 32 patients (8.3%) had CHIKV infections. Compared with real-time polymerase chain reaction, microscopy demonstrated a percent positive agreement of 49.7%. The percentage of malaria cases detected by microscopy varied significantly between clinical sites. Arboviral infections were the clinical diagnosis for patients on the Indian Ocean coast (91 of 238, 38.2%) significantly more often than patients in the Lake Victoria region (2 of 145, 1.4%; P < .001). However, detection of CHIKV infections was significantly higher in the Lake Victoria region (19 of 145 [13.1%] vs 13 of 239 [5.4%]; P = .012).The clinical diagnosis of patients with an acute febrile illness, even when aided by microscopy, remains inaccurate in malaria-endemic areas, contributing to inappropriate management decisions.

  View details for PubMedID 28702473

• Metagenomic DNA Sequencing for the Diagnosis of Intraocular Infections. Ophthalmology Doan, T., Acharya, N. R., Pinsky, B. A., Sahoo, M. K., Chow, E. D., Banaei, N., Budvytiene, I., Cevallos, V., Zhong, L., Zhou, Z., Lietman, T. M., DeRisi, J. L. 2017

  View details for DOI 10.1016/j.ophtha.2017.03.045

  View details for PubMedID 28526549

• Susceptibility of Candida albicans from Cystic Fibrosis Patients. Mycopathologia Sabino, R., Carolino, E., Moss, R. B., Banaei, N., Verissimo, C., Stevens, D. A. 2017

  Abstract

  Candida albicans is a common microbe, colonizer and potential pathogen found in respiratory cultures of cystic fibrosis (CF) patients. Because of possible development of resistance in patient isolates resulting from residence in the abnormal milieu of CF patient airways, or from exposure to antifungals, and considering the possibility of patient-to-patient spread of microbes and reports of elevated resistance to other fungal pathogens, it was important to assay the susceptibility of isolates of Candida and compare that profile to isolates from the community. In our center, and unlike another fungal pathogen, no increase in resistance of Candida isolates of the CF cohort was found.

  View details for DOI 10.1007/s11046-017-0133-9

  View details for PubMedID 28421452

• Integrated Biosensor Assay for Rapid Uropathogen Identification and Phenotypic Antimicrobial Susceptibility Testing. European urology focus Altobelli, E., Mohan, R., Mach, K. E., Sin, M. L., Anikst, V., Buscarini, M., Wong, P. K., Gau, V., Banaei, N., Liao, J. C. 2017; 3 (2-3): 293?99

  Abstract

  BACKGROUND: Standard diagnosis of urinary tract infection (UTI) via urine culture for pathogen identification (ID) and antimicrobial susceptibility testing (AST) takes 2-3 d. This delay results in empiric treatment and contributes to the misuse of antibiotics and the rise of resistant pathogens. A rapid diagnostic test for UTI may improve patient care and antibiotic stewardship.OBJECTIVE: To develop and validate an integrated biosensor assay for UTI diagnosis, including pathogen ID and AST, with determination of the minimum inhibitory concentration (MIC) for ciprofloxacin.DESIGN, SETTING, AND PARTICIPANTS: Urine samples positive for Enterobacteriaceae (n=84) or culture-negative (n=23) were obtained from the Stanford Clinical Microbiology Laboratory between November 2013 and September 2014. Each sample was diluted and cultured for 5h with and without ciprofloxacin, followed by quantitative detection of bacterial 16S rRNA using a single electrochemical biosensor array functionalized with a panel of complementary DNA probes. Pathogen ID was determined using universal bacterial, Enterobacteriaceae (EB), and pathogen-specific probes. Phenotypic AST with ciprofloxacin MIC was determined using an EB probe to measure 16S rRNA levels as a function of bacterial growth.MEASUREMENTS: Electrochemical signals for pathogen ID at 6 SD over background were considered positive. An MIC signal of 0.4 log units lower than the no-antibiotic control indicated sensitivity. Results were compared to clinical microbiology reports.RESULTS AND LIMITATIONS: For pathogen ID, the assay had 98.5% sensitivity, 96.6% specificity, 93.0% positive predictive value, and 99.3% negative predictive value. For ciprofloxacin MIC the categorical and essential agreement was 97.6%. Further automation, testing of additional pathogens and antibiotics, and a full prospective study will be necessary for translation to clinical use.CONCLUSIONS: The integrated biosensor platform achieved microbiological results including MIC comparable to standard culture in a significantly shorter assay time. Further assay automation will allow clinical translation for rapid molecular diagnosis of UTI.PATIENT SUMMARY: We have developed and validated a biosensor test for rapid diagnosis of urinary tract infections. Clinical translation of this device has the potential to significantly expedite and improve treatment of urinary tract infections.

  View details for PubMedID 28753748

• Clostridium difficile rates in asymptomatic and symptomatic hospitalized patients using nucleic acid testing. Diagnostic microbiology and infectious disease Truong, C., Schroeder, L. F., Gaur, R., Anikst, V. E., Komo, I., Watters, C., McCalley, E., Kulik, C., Pickham, D., Lee, N. J., Banaei, N. 2017; 87 (4): 365-370

  Abstract

  The Clostridium difficile rate in symptomatic patients represents both those with C. difficile infection (CDI) and those with colonization. To predict the extent of CDI overdiagnosis, we compared the asymptomatic colonization rate to the symptomatic positivity rate in hospitalized patients using nucleic acid testing.Between July 2014 and April 2015, formed stool samples were collected from asymptomatic patients after admission to 3 hospital wards at the Stanford Hospital. Stool samples from symptomatic patients with suspected CDI in the same wards were collected for testing per provider order. The GeneXpert C. difficile tcdB polymerase chain reaction (PCR) assay (Cepheid, Sunnyvale, CA, USA) was performed on all stool samples and PCR cycle threshold was used as a measure of genomic equivalents. Chart review was performed to obtain clinical history and medication exposure.We found an asymptomatic C. difficile carriage rate of 11.8% (43/365) (95% confidence interval [CI], 8.5-15.1%) and a positivity rate in symptomatic patients of 15.4% (54/351) (95% CI, 11.6-19.2%; P=0.19). The median PCR cycle thresholds was not significantly different between asymptomatic carriers and symptomatic positives (29.5 versus 27.3; P=0.07). Among asymptomatic patients, 11.6% (5/43) of carriers and 8.4% (27/322; P=0.56) of noncarriers subsequently became symptomatic CDI suspects within the same hospitalization. Single and multivariate analysis did not identify any demographic or clinical factors as being significantly associated with C. difficile carriage.Asymptomatic C. difficile carriage rate was similar to symptomatic positivity rate. This suggests the majority of PCR-positive results in symptomatic patients are likely due to C. difficile colonization. Disease-specific biomarkers are needed to accurately diagnose patients with C. difficile disease.

  View details for DOI 10.1016/j.diagmicrobio.2016.12.014

  View details for PubMedID 28087170

• Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas MYCOPATHOLOGIA Nazik, H., Moss, R. B., Karna, V., Clemons, K. V., Banaei, N., Cohen, K., Choudhary, V., Stevens, D. A. 2017; 182 (3-4): 315-318

  View details for DOI 10.1007/s11046-016-0087-3

  View details for Web of Science ID 000397206000005

• New and developing diagnostic technologies for urinary tract infections. Nature reviews. Urology Davenport, M., Mach, K. E., Shortliffe, L. M., Banaei, N., Wang, T., Liao, J. C. 2017

  Abstract

  Timely and accurate identification and determination of the antimicrobial susceptibility of uropathogens is central to the management of UTIs. Urine dipsticks are fast and amenable to point-of-care testing, but do not have adequate diagnostic accuracy or provide microbiological diagnosis. Urine culture with antimicrobial susceptibility testing takes 2-3 days and requires a clinical laboratory. The common use of empirical antibiotics has contributed to the rise of multidrug-resistant organisms, reducing treatment options and increasing costs. In addition to improved antimicrobial stewardship and the development of new antimicrobials, novel diagnostics are needed for timely microbial identification and determination of antimicrobial susceptibilities. New diagnostic platforms, including nucleic acid tests and mass spectrometry, have been approved for clinical use and have improved the speed and accuracy of pathogen identification from primary cultures. Optimization for direct urine testing would reduce the time to diagnosis, yet these technologies do not provide comprehensive information on antimicrobial susceptibility. Emerging technologies including biosensors, microfluidics, and other integrated platforms could improve UTI diagnosis via direct pathogen detection from urine samples, rapid antimicrobial susceptibility testing, and point-of-care testing. Successful development and implementation of these technologies has the potential to usher in an era of precision medicine to improve patient care and public health.

  View details for DOI 10.1038/nrurol.2017.20

  View details for PubMedID 28248946

• Infection Rates. Journal of clinical microbiology Truong, C. Y., Gombar, S., Wilson, R., Sundararajan, G., Tekic, N., Holubar, M., Shepard, J., Madison, A., Tompkins, L., Shah, N., Deresinski, S., Schroeder, L. F., Banaei, N. 2017

  Abstract

  Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [?3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days (P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days (P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days (P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates.

  View details for DOI 10.1128/JCM.02319-16

  View details for PubMedID 28250001

• Is Follow-up Testing with FilmArray Gastrointestinal Multiplex PCR Panel Necessary? Journal of clinical microbiology Park, S., Hitchcock, M. M., Gomez, C. A., Banaei, N. 2017

  Abstract

  FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT) is a simple, sample-to-answer, on-demand, multiplex, nucleic acid amplification test for syndromic diagnosis of infectious gastroenteritis. The aim of this study was to measure the yield of follow-up testing with FilmArray GI Panel within 4 weeks of an initial test. Consecutive adult and pediatric patients tested at an academic institution between August 2015 and June 2016 were included in this study. Of 145 follow-up tests in 106 unique patients with an initial negative result, 134 (92.4%) tests and 98 (92.5%) patients remained negative upon follow-up testing. Excluding targets that are not reported at our institution (Clostridium difficile, enteroaggregative Escherichia coli, enteropathogenic E. coli, and enterotoxigenic E. coli), 137 (94.5%) follow-up tests and 101 (95.3%) patients remained negative. Weekly conversion rates were not significantly different across the 4-week follow-up interval. No epidemiological or clinical factors were significantly associated with a negative to positive conversion. Of 80 follow-up tests in patients with an initial positive result, 43 (53.8%) remained positive for the same target, 34 (42.5%) were negative, and 3 were positive for a different target (3.8%). Follow-up testing with FilmArray GI Panel within 4 weeks of a negative result rarely changed the initial result, and the follow-up test reverted to negative less than half the time after an initial positive result. In the absence of clinical or epidemiological evidence for a new infection, follow-up testing should be limited and FilmArray GI Panel should not be used as a test of cure.

  View details for DOI 10.1128/JCM.02354-16

  View details for PubMedID 28122874

• Delayed Diagnosis of Tuberculous Meningitis Misdiagnosed as Herpes Simplex Virus-1 Encephalitis With the FilmArray Syndromic Polymerase Chain Reaction Panel. Open forum infectious diseases Gomez, C. A., Pinsky, B. A., Liu, A., Banaei, N. 2017; 4 (1): ofw245-?

  Abstract

  The FilmArray meningitis/encephalitis (ME) panel is a novel syndromic, nucleic acid amplification test for diagnosis of acute meningitis and encephalitis. Emerging data on its performance are concerning for false-positive results. We present a case of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray ME panel. Strategies to mitigate erroneous results are discussed.

  View details for DOI 10.1093/ofid/ofw245

  View details for PubMedID 28540320

• Intramolecular substitution uncages fluorogenic probes for detection of metallo-carbapenemase-expressing bacteria. Chemical science Song, A., Cheng, Y., Xie, J., Banaei, N., Rao, J. 2017; 8 (11): 7669?74

  Abstract

  This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of ?-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group-dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.

  View details for PubMedID 29568429

• Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Gomez, C. A., Budvytiene, I., Zemek, A. J., Banaei, N. 2017; 65 (12): 2035?41

  Abstract

  Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD.Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified.Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases.The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

  View details for PubMedID 29020284

• Intramolecular substitution uncages fluorogenic probes for detection of metallo-carbapenemase-expressing bacteria Chemical Science Song, A., Cheng, Y., Xie, J., Banaei, N., Rao, J. 2017; 8 (11): 7669-7674

  Abstract

  This work reports a novel caging strategy for designing fluorogenic probes to detect the activity of ?-lactamases. The caging strategy uses a thiophenyl linker connected to a fluorophore caged by a good leaving group-dinitrophenyl. The uncaging proceeds in two steps through the sulfa-releasing and subsequent intramolecular substitution. The length of the linker has been examined and optimized to maximize the rate of intramolecular reaction and thus the rate of fluorescence activation. Finally based on this strategy, we prepared a green fluorogenic probe CAT-7 and validated its selectivity for detecting metallo-carbapenemases (VIM-27, IMP-1, NDM-1) in carbapenem-resistant Enterobacteriaceae (CRE) lysates.

  View details for DOI 10.1039/C7SC02416A

  View details for PubMedCentralID PMC5849144

• Determining the cause of recurrent Clostridium difficile infection using whole genome sequencing DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Sim, J. H., Truong, C., Minot, S. S., Greenfield, N., Budvytiene, I., Lohith, A., Anikst, V., Pourmand, N., Banaei, N. 2017; 87 (1): 11-16

  View details for DOI 10.1016/j.diagmicrobio.2016.09.023

  View details for Web of Science ID 000390183100003

• Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm. Mycopathologia Anand, R., Moss, R. B., Sass, G., Banaei, N., Clemons, K. V., Martinez, M., Stevens, D. A. 2017

  Abstract

  Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

  View details for PubMedID 28785939

• Susceptibility of Candida albicans from Cystic Fibrosis Patients Mycopathologica Sabino, R., Carolino, E., Moss, R. B., Banaei, N., Verissimo, C., Stevens, D. A. 2017

  Abstract

  Candida albicans is a common microbe, colonizer and potential pathogen found in respiratory cultures of cystic fibrosis (CF) patients. Because of possible development of resistance in patient isolates resulting from residence in the abnormal milieu of CF patient airways, or from exposure to antifungals, and considering the possibility of patient-to-patient spread of microbes and reports of elevated resistance to other fungal pathogens, it was important to assay the susceptibility of isolates of Candida and compare that profile to isolates from the community. In our center, and unlike another fungal pathogen, no increase in resistance of Candida isolates of the CF cohort was found.

  View details for DOI 10.1007/s11046-017-0133-9

• Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease Clinical Infectious Diseases Gomez, C. A., Budvytiene, I., Zemek, A., Banaei, N. 2017: 2035?41

  Abstract

  Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD.Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified.Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases.The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

  View details for DOI 10.1093/cid/cix728

• Are Cystic Fibrosis Aspergillus fumigatus Isolates Different? Intermicrobial Interactions with Pseudomonas. Mycopathologia Nazik, H., Moss, R. B., Karna, V., Clemons, K. V., Banaei, N., Cohen, K., Choudhary, V., Stevens, D. A. 2016: -?

  Abstract

  Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.

  View details for PubMedID 27822731

• Adenosine triphosphate bioluminescence for bacteriological surveillance and reprocessing strategies for minimizing risk of infection transmission by duodenoscopes. Gastrointestinal endoscopy Sethi, S., Huang, R. J., Barakat, M. T., Banaei, N., Friedland, S., Banerjee, S. 2016

  Abstract

  Recent outbreaks of duodenoscope-transmitted infections underscore the importance of adequate endoscope reprocessing. Adenosine triphosphate (ATP) bioluminescence testing allows rapid evaluation of endoscopes for bacteriologic/biologic residue. In this prospective study we evaluate the utility of ATP in bacteriologic surveillance and the effects of endoscopy staff education and dual cycles of cleaning and high-level disinfection (HLD) on endoscope reprocessing.ATP bioluminescence was measured after precleaning, manual cleaning, and HLD on rinsates from suction-biopsy channels of all endoscopes and elevator channels of duodenoscopes/linear echoendoscopes after use. ATP bioluminescence was remeasured in duodenoscopes (1) after re-education and competency testing of endoscopy staff and subsequently (2) after 2 cycles of precleaning and manual cleaning and single cycle of HLD or (3) after 2 cycles of precleaning, manual cleaning, and HLD.The ideal ATP bioluminescence benchmark of <200 relative light units (RLUs) after manual cleaning was achieved from suction-biopsy channel rinsates of all endoscopes, but 9 of 10 duodenoscope elevator channel rinsates failed to meet this benchmark. Re-education reduced RLUs in duodenoscope elevator channel rinsates after precleaning (23,218.0 vs 1340.5 RLUs, P < .01) and HLD (177.0 vs 12.0 RLUs, P < .01). After 2 cycles of manual cleaning/HLD, duodenoscope elevator channel RLUs achieved levels similar to sterile water, with corresponding negative cultures.ATP testing offers a rapid, inexpensive alternative for detection of endoscope microbial residue. Re-education of endoscopy staff and 2 cycles of cleaning and HLD decreased elevator channel RLUs to levels similar to sterile water and may therefore minimize the risk of transmission of infections by duodenoscopes.

  View details for DOI 10.1016/j.gie.2016.10.035

  View details for PubMedID 27818222

• Determining the cause of recurrent Clostridium difficile infection using whole genome sequencing. Diagnostic microbiology and infectious disease Sim, J. H., Truong, C., Minot, S. S., Greenfield, N., Budvytiene, I., Lohith, A., Anikst, V., Pourmand, N., Banaei, N. 2016

  Abstract

  Understanding the contribution of relapse and reinfection to recurrent Clostridium difficile infection (CDI) has implications for therapy and infection prevention, respectively. We used whole genome sequencing to determine the relation of C. difficile strains isolated from patients with recurrent CDI at an academic medical center in the United States. Thirty-five toxigenic C. difficile isolates from 16 patients with 19 recurrent CDI episodes with median time of 53.5days (range, 13-362) between episodes were whole genome sequenced on the Illumina MiSeq platform. In 84% (16) of recurrences, the cause of recurrence was relapse with prior strain of C. difficile. In 16% (3) of recurrent episodes, reinfection with a new strain of C. difficile was the cause. In conclusion, the majority of CDI recurrences at our institution were due to infection with the same strain rather than infection with a new strain.

  View details for DOI 10.1016/j.diagmicrobio.2016.09.023

  View details for PubMedID 27771207

• Serial testing for latent tuberculosis using QuantiFERON-TB Gold In-Tube: A Markov model SCIENTIFIC REPORTS Moses, M. W., Zwerling, A., Cattamanchi, A., Denkinger, C. M., Banaei, N., Kik, S. V., Metcalfe, J., Pai, M., Dowdy, D. 2016; 6

  Abstract

  Healthcare workers (HCWs) in low-incidence settings are often serially tested for latent TB infection (LTBI) with the QuantiFERON-TB Gold In-Tube (QFT) assay, which exhibits frequent conversions and reversions. The clinical impact of such variability on serial testing remains unknown. We used a microsimulation Markov model that accounts for major sources of variability to project diagnostic outcomes in a simulated North American HCW cohort. Serial testing using a single QFT with the recommended conversion cutoff (IFN-g?>?0.35?IU/mL) resulted in 24.6% (95% uncertainty range, UR: 23.8-25.5) of the entire population testing false-positive over ten years. Raising the cutoff to >1.0?IU/mL or confirming initial positive results with a (presumed independent) second test reduced this false-positive percentage to 2.3% (95%UR: 2.0-2.6%) or 4.1% (95%UR: 3.7-4.5%), but also reduced the proportion of true incident infections detected within the first year of infection from 76.5% (95%UR: 66.3-84.6%) to 54.8% (95%UR: 44.6-64.5%) or 61.5% (95%UR: 51.6-70.9%), respectively. Serial QFT testing of HCWs in North America may result in tremendous over-diagnosis and over-treatment of LTBI, with nearly thirty false-positives for every true infection diagnosed. Using higher cutoffs for conversion or confirmatory tests (for initial positives) can mitigate these effects, but will also diagnose fewer true infections.

  View details for DOI 10.1038/srep30781

  View details for Web of Science ID 000380659100001

  View details for PubMedID 27469388

  View details for PubMedCentralID PMC4965809

• Rapid Diagnosis of Tuberculosis from Analysis of Urine Volatile Organic Compounds ACS SENSORS Lim, S. H., Martino, R., Anikst, V., Xu, Z., Mix, S., Benjamin, R., Schub, H., Eiden, M., Rhodes, P. A., Banaei, N. 2016; 1 (7): 852-856

  View details for DOI 10.1021/acssensors.6b00309

  View details for Web of Science ID 000385464900005

• Organism burden, toxin concentration, and lactoferrin concentration do not distinguish between clinically significant and nonsignificant diarrhea in patients with Clostridium difficile DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Anikst, V. E., Gaur, R. L., Schroeder, L. F., Banaei, N. 2016; 84 (4): 343-346

  Abstract

  Clostridium difficile infection is often overdiagnosed in patients with mild diarrhea. We evaluated 4 biomarkers as surrogates for clinically significant diarrhea (?3 episodes in 24hours) in 59 PCR-positive patients with and 59 PCR-positive patients without clinically significant diarrhea. Organism burden (median tcdB cycle threshold value, 26.9 versus 27.1, P=0.25) and toxin A and B concentrations (toxin A, median, 0 versus 0ng/mL, P=0.42; toxin B, median, 0 versus 0ng/mL, P=0.25) were not significantly different between patients with and without clinically significant diarrhea. Fecal lactoferrin concentrations were significantly increased in patients with clinically significant diarrhea (median, 99.0 versus 55.1?g/mL, P=0.05); however, lactoferrin could not sufficiently classify patients into those with and without clinically significant diarrhea. Interventions that limit C. difficile testing to patients with clinically significant diarrhea are needed to improve the positive predictive value of C. difficile diagnostics.

  View details for DOI 10.1016/j.diagmicrobio.2015.11.022

  View details for Web of Science ID 000372768800014

• Interferon Gamma Release Assays for Latent Tuberculosis: What Are the Sources of Variability? Journal of clinical microbiology Banaei, N., Gaur, R. L., Pai, M. 2016; 54 (4): 845-850

  Abstract

  Interferon-? release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI). IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies in low risk individuals have revealed higher false conversion rates with IGRAs compared with TST. Reproducibility studies have identified various sources of variability that contribute to non-reproducible results. Sources of variability can be broadly classified as pre-analytical, analytical, post-analytical, manufacturing, and immunological. In this review, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability.

  View details for DOI 10.1128/JCM.02803-15

  View details for PubMedID 26763969

• Bacterial culture detection and identification in blood agar plates with an optoelectronic nose. Analyst Lim, S. H., Mix, S., Anikst, V., Budvytiene, I., Eiden, M., Churi, Y., Queralto, N., Berliner, A., Martino, R. A., Rhodes, P. A., Banaei, N. 2016; 141 (3): 918-925

  Abstract

  Clinical microbiology automation is currently limited by the lack of an in-plate culture identification system. Using an inexpensive, printed, disposable colorimetric sensor array (CSA) responsive to the volatiles emitted into plate headspace by microorganisms during growth, we report here that not only the presence but the species of bacteria growing in plate was identified before colonies are visible. In 1894 trials, 15 pathogenic bacterial species cultured on blood agar were identified with 91.0% sensitivity and 99.4% specificity within 3 hours of detection. The results indicate CSAs integrated into Petri dish lids present a novel paradigm to speciate microorganisms, well-suited to integration into automated plate handling systems.

  View details for DOI 10.1039/c5an01990g

  View details for PubMedID 26753182

• Toxin Immunoassays and Clostridium difficile Infection. JAMA internal medicine Banaei, N., Schroeder, L. F. 2016; 176 (3): 413

  View details for PubMedID 26954049

• Delayed Diagnosis of Tuberculous Meningitis Misdiagnosed as Herpes Simplex Virus-1 Encephalitis With the FilmArray Syndromic Polymerase Chain Reaction Panel Open Forum Infectious Diseases Gomez, C. A., Pinsky, B. A., Liu, A., Banaei, N. 2016: ofw245

  Abstract

  The FilmArray meningitis/encephalitis (ME) panel is a novel syndromic, nucleic acid amplification test for diagnosis of acute meningitis and encephalitis. Emerging data on its performance are concerning for false-positive results. We present a case of tuberculous meningitis misdiagnosed as herpes simplex virus-1 encephalitis with the FilmArray ME panel. Strategies to mitigate erroneous results are discussed.

  View details for DOI 10.1093/ofid/ofw245

  View details for PubMedCentralID PMC5437853

• Rapid Diagnosis of Tuberculosis from Analysis of Urine Volatile Organic Compounds. ACS sensors Lim, S. H., Martino, R., Anikst, V., Xu, Z., Mix, S., Benjamin, R., Schub, H., Eiden, M., Rhodes, P. A., Banaei, N. 2016; 1 (7): 852?56

  Abstract

  The World Health Organization has called for simple, sensitive, and non-sputum diagnostics for tuberculosis. We report development of a urine tuberculosis test using a colorimetric sensor array (CSA). The sensor comprised of 73 different indicators captures high-dimensional, spatiotemporal signatures of volatile chemicals emitted by human urine samples. The sensor responses to 63 urine samples collected from 22 tuberculosis cases and 41 symptomatic controls were measured under five different urine test conditions. Basified testing condition yielded the best accuracy with 85.5% sensitivity and 79.5% specificity. The CSA urine assay offers desired features needed for tuberculosis diagnosis in endemic settings.

  View details for PubMedID 29057329

• First case of infectious endocarditis caused by Parvimonas micra. Anaerobe Gomez, C. A., Gerber, D. A., Zambrano, E., Banaei, N., Deresinski, S., Blackburn, B. G. 2015; 36: 53-55

  Abstract

  P. micra is an anaerobic Gram-positive cocci, and a known commensal organism of the human oral cavity and gastrointestinal tract. Although it has been classically described in association with endodontic disease and peritonsillar infection, recent reports have highlighted the role of P. micra as the primary pathogen in the setting of invasive infections. In its most recent taxonomic classification, P. micra has never been reported causing infectious endocarditis in humans. Here, we describe a 71 year-old man who developed severe native valve endocarditis complicated by aortic valvular destruction and perivalvular abscess, requiring emergent surgical intervention. Molecular sequencing enabled identification of P. micra.

  View details for DOI 10.1016/j.anaerobe.2015.10.007

  View details for PubMedID 26485192

• Next-Generation Sequencing for Infectious Disease Diagnosis and Management A Report of the Association for Molecular Pathology JOURNAL OF MOLECULAR DIAGNOSTICS Lefterova, M. I., Suarez, C. J., Banaei, N., Pinsky, B. A. 2015; 17 (6): 623-634

  Abstract

  Next-generation sequencing (NGS) technologies are increasingly being used for diagnosis and monitoring of infectious diseases. Herein, we review the application of NGS in clinical microbiology, focusing on genotypic resistance testing, direct detection of unknown disease-associated pathogens in clinical specimens, investigation of microbial population diversity in the human host, and strain typing. We have organized the review into three main sections: i) applications in clinical virology, ii) applications in clinical bacteriology, mycobacteriology, and mycology, and iii) validation, quality control, and maintenance of proficiency. Although NGS holds enormous promise for clinical infectious disease testing, many challenges remain, including automation, standardizing technical protocols and bioinformatics pipelines, improving reference databases, establishing proficiency testing and quality control measures, and reducing cost and turnaround time, all of which would be necessary for widespread adoption of NGS in clinical microbiology laboratories.

  View details for DOI 10.1016/j.jmoldx.2015.07.004

  View details for Web of Science ID 000363830000001

  View details for PubMedID 26433313

• Molecular Testing for Plasmodium falciparum by Use of Serum or Plasma and Comparison with Microscopy and Rapid Diagnostic Testing in Febrile Nigerian Patients. Journal of clinical microbiology Waggoner, J. J., Okangba, C., Mohamed-Hadley, A., Lefterova, M. I., Banaei, N., Oyibo, W., Pinsky, B. A. 2015; 53 (11): 3596-3600

  Abstract

  Plasmodium nucleic acids have been detected in serum and plasma, but there is little published data describing the diagnostic performance of malaria nucleic acid amplification tests (NAATs) using these specimen types. Previously, our group described a multiplex NAAT for the detection of dengue virus, Leptospira, and Plasmodium species with a callout for P. falciparum (the DLM assay) that demonstrated sensitive detection of P. falciparum from plasma samples during initial evaluation. In this study, we evaluated the sensitivity and specificity of P. falciparum detection in febrile Nigerian patients using the DLM assay, microscopy, and a rapid diagnostic test (BinaxNOW Malaria). Assay performances were compared using a composite reference, which was considered positive if malaria was detected by two or more methods. Serum (n = 182) or plasma (n = 148) from 317 patients was tested; the average sample volume was 70 ?l (range, 5 to 300 ?l). The sensitivity and specificity of the DLM assay were 97.1% and 93.5%, respectively. The sensitivity of the malaria rapid diagnostic test (98.1%) was similar to that of the DLM assay, and both proved significantly more sensitive than microscopy (79%; P < 0.0001). When analysis was limited to samples with ?75 ?l of serum or plasma, the sensitivity of the DLM assay improved to 99% and specificity was 97.5%. For P. falciparum cases, cycle threshold values in the DLM assay correlated with the parasite density detected by microscopy (Spearman's rank correlation coefficient, P < 0.0001). In conclusion, malaria detection using the DLM assay on serum or plasma is more sensitive than and equal in specificity to microscopy in patients with P. falciparum malaria.

  View details for DOI 10.1128/JCM.01876-15

  View details for PubMedID 26354810

  View details for PubMedCentralID PMC4609704

• First case of mesh infection due to Coccidioides spp. and literature review of fungal mesh infections after hernia repair. Mycoses Forrester, J. D., Gomez, C. A., Forrester, J. A., Nguyen, M., Gregg, D., Deresinski, S., Banaei, N., Weiser, T. G. 2015; 58 (10): 582-587

  Abstract

  Fungal mesh infections are a rare complication of hernia repairs with mesh. The first case of Coccidioides spp. mesh infection is described, and a systematic literature review of all known fungal mesh infections was performed. Nine cases of fungal mesh infection are reviewed. Female and male patients are equally represented, median age is 49.5 years, and critical illness and preinfection antibiotic use were common. Fungal mesh infections are rare, but potentially fatal, complications of hernias repaired with mesh.

  View details for DOI 10.1111/myc.12364

  View details for PubMedID 26293423

• A small-molecule antivirulence agent for treating Clostridium difficile infection. Science translational medicine Bender, K. O., Garland, M., Ferreyra, J. A., Hryckowian, A. J., Child, M. A., Puri, A. W., Solow-Cordero, D. E., Higginbottom, S. K., Segal, E., Banaei, N., Shen, A., Sonnenburg, J. L., Bogyo, M. 2015; 7 (306): 306ra148-?

  Abstract

  Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.

  View details for DOI 10.1126/scitranslmed.aac9103

  View details for PubMedID 26400909

• A small-molecule antivirulence agent for treating Clostridium difficile infection SCIENCE TRANSLATIONAL MEDICINE Bender, K. O., Garland, M., Ferreyra, J. A., Hryckowian, A. J., Child, M. A., Puri, A. W., Solow-Cordero, D. E., Higginbottom, S. K., Segal, E., Banaei, N., Shen, A., Sonnenburg, J. L., Bogyo, M. 2015; 7 (306)

  Abstract

  Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.

  View details for DOI 10.1126/scitranslmed.aac9103

  View details for Web of Science ID 000365232600002

  View details for PubMedID 26400909

• In vitro immunomodulation for enhancing T cell-based diagnosis of Mycobacterium tuberculosis infection DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Slater, M., Minh-Chi Tran, M. C., Platt, L., Luu, L. T., Phan, H. T., Pham, P. T., Do, T. B., Nguyen, H. T., Gaur, R. L., Parsonnet, J., Cattamanchi, A., Luo, R., Nahid, P., Banaei, N. 2015; 83 (1): 41-45

  Abstract

  Interferon-gamma release assays have limited sensitivity for detecting latent tuberculosis infection. In this study, we determine if the addition of immunomodulators to the QuantiFERON-TB Gold In-Tube (QFT-GIT) increased test sensitivity without compromising specificity. We prospectively compared QFT-GIT results with and without incubation with 2 immunomodulators (lipopolysaccharide [LPS] and polyinosine-polycytidylic acid [PolyIC]) in 2 cohorts-113 culture-confirmed tuberculosis (TB) subjects in Hanoi, Vietnam, and 226 documented QFT-GIT-negative, low TB risk health care workers undergoing annual TB screening at a US academic institution. Sensitivity of the tests in TB subjects was 84.1% with the standard QFT-GIT and 85.8% and 74.3% after incubation with LPS and PolyIC, respectively. Specificity in low TB risk health care workers was 100% with the standard QFT-GIT by design and 86.7% with LPS and 63.3% with PolyIC. In conclusion, use of the 2 immunomodulators did not improve sensitivity of the QFT-GIT in TB patients and reduced specificity in low-risk health care workers.

  View details for DOI 10.1016/j.diagmicrobio.2015.05.007

  View details for Web of Science ID 000359754000010

  View details for PubMedID 26081239

• Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium PLOS ONE Ferreira, J. A., Penner, J. C., Moss, R. B., Haagensen, J. A., Clemons, K. V., Spormann, A. M., Nazik, H., Cohen, K., Banaei, N., Carolino, E., Stevens, D. A. 2015; 10 (8)

  View details for DOI 10.1371/journal.pone.0134692

  View details for Web of Science ID 000359121100068

  View details for PubMedID 26252384

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient JOURNAL OF CLINICAL MICROBIOLOGY Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015; 53 (8): 2749-2752

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for DOI 10.1128/JCM.00834-15

  View details for Web of Science ID 000358290200055

• Rapid Detection of Acquired and Inducible Clarithromycin Resistance in Mycobacterium abscessus Group by a Simple Real-Time PCR Assay JOURNAL OF CLINICAL MICROBIOLOGY Luo, R. F., Curry, C., Taylor, N., Budvytiene, I., Banaei, N. 2015; 53 (7): 2337-2339

  Abstract

  By targeting the erm(41) and rrl genes in the Mycobacterium abscessus group, a multiplex real-time PCR assay for clarithromycin resistance showed 95% (38/40) concordance with nucleic acid testing and 95% (37/39) concordance with phenotypic testing. This assay provides a simple and rapid alternative to extended incubation or erm(41) sequencing.

  View details for DOI 10.1128/JCM.00132-15

  View details for Web of Science ID 000358287700046

  View details for PubMedCentralID PMC4473208

• Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing JOURNAL OF CLINICAL MICROBIOLOGY Sim, J. H., Anikst, V., Lohith, A., Pourmand, N., Banaei, N. 2015; 53 (7): 2329-2331

  Abstract

  Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform.

  View details for DOI 10.1128/JCM.00956-15

  View details for Web of Science ID 000358287700044

  View details for PubMedCentralID PMC4473243

• Molecular epidemiology of Aspergillus collected from cystic fibrosis patients. Journal of cystic fibrosis Sabino, R., Ferreira, J. A., Moss, R. B., Valente, J., Veríssimo, C., Carolino, E., Clemons, K. V., Everson, C., Banaei, N., Penner, J., Stevens, D. A. 2015; 14 (4): 474-481

  Abstract

  Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease.Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), ?-tubulin, and calmodulin sequencing.Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found.To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

  View details for DOI 10.1016/j.jcf.2014.10.005

  View details for PubMedID 25459562

• Molecular epidemiology of Aspergillus collected from cystic fibrosis patients JOURNAL OF CYSTIC FIBROSIS Sabino, R., Ferreira, J. A., Moss, R. B., Valente, J., Verissimo, C., Carolino, E., Clemons, K. V., Everson, C., Banaei, N., Penner, J., Stevens, D. A. 2015; 14 (4): 474-481

  View details for DOI 10.1016/j.jcf.2014.10.005

  View details for Web of Science ID 000358818800009

• Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood JOURNAL OF CLINICAL MICROBIOLOGY Lefterova, M. I., Budvytiene, I., Sandlund, J., Faernert, A., Banaei, N. 2015; 53 (7): 2251-2257

  Abstract

  Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

  View details for DOI 10.1128/JCM.00542-15

  View details for Web of Science ID 000358287700034

  View details for PubMedCentralID PMC4473218

• Burden of Clostridium difficile infection in the United States. New England journal of medicine Banaei, N., Anikst, V., Schroeder, L. F. 2015; 372 (24): 2368-2369

  View details for DOI 10.1056/NEJMc1505190#SA2

  View details for PubMedID 26061852

• Ribosomal RNA gene sequencing for early diagnosis of Blastomyces dermatitidis infection. International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases Morjaria, S., Otto, C., Moreira, A., Chung, R., Hatzoglou, V., Pillai, M., Banaei, N., Tang, Y. W., Figueroa, C. J. 2015; 37: 122?24

  Abstract

  Prompt detection and identification of fungal pathogens at the genus and species level is critical in order to provide timely antifungal therapy. Here, we highlight the vital role of molecular diagnostics in achieving a fast and definitive diagnosis of disseminated blastomycosis in a diabetic patient presenting as a brain mass initially thought to be tumoral in nature. A broad-range PCR amplification and sequencing of the fungal ribosomal RNA genes on brain biopsy tissue obtained during elective craniotomy revealed a final microbial identification of Ajellomyces dermatitidis (telemorph of Blastomyces dermatitidis).

  View details for PubMedID 26129971

• Fatal West Nile Virus Encephalitis in a Heart Transplant Recipient. Journal of clinical microbiology Gomez, A. J., Waggoner, J. J., Itoh, M., Hollander, S. A., Gutierrez, K. M., Budvytiene, I., Banaei, N., Pinsky, B. A. 2015

  Abstract

  The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

  View details for PubMedID 25994169

• Inhibition of Aspergillus fumigatus and Its Biofilm by Pseudomonas aeruginosa Is Dependent on the Source, Phenotype and Growth Conditions of the Bacterium. PloS one Ferreira, J. A., Penner, J. C., Moss, R. B., Haagensen, J. A., Clemons, K. V., Spormann, A. M., Nazik, H., Cohen, K., Banaei, N., Carolino, E., Stevens, D. A. 2015; 10 (8)

  Abstract

  Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

  View details for DOI 10.1371/journal.pone.0134692

  View details for PubMedID 26252384

  View details for PubMedCentralID PMC4529298

• Optimized Protocol for Simple Extraction of High-Quality Genomic DNA from Clostridium difficile for Whole-Genome Sequencing. Journal of clinical microbiology Sim, J. H., Anikst, V., Lohith, A., Pourmand, N., Banaei, N. 2015; 53 (7): 2329?31

  Abstract

  Successful sequencing of the Clostridium difficile genome requires high-quality genomic DNA (gDNA) as the starting material. gDNA extraction using conventional methods is laborious. We describe here an optimized method for the simple extraction of C. difficile gDNA using the QIAamp DNA minikit, which yielded high-quality sequence reads on the Illumina MiSeq platform.

  View details for PubMedID 25878343

  View details for PubMedCentralID PMC4473243

• Simple Real-Time PCR and Amplicon Sequencing Method for Identification of Plasmodium Species in Human Whole Blood. Journal of clinical microbiology Lefterova, M. I., Budvytiene, I., Sandlund, J., Färnert, A., Banaei, N. 2015; 53 (7): 2251?57

  Abstract

  Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

  View details for PubMedID 25972416

  View details for PubMedCentralID PMC4473218

• Rapid Detection of Acquired and Inducible Clarithromycin Resistance in Mycobacterium abscessus Group by a Simple Real-Time PCR Assay. Journal of clinical microbiology Luo, R. F., Curry, C., Taylor, N., Budvytiene, I., Banaei, N. 2015; 53 (7): 2337?39

  Abstract

  By targeting the erm(41) and rrl genes in the Mycobacterium abscessus group, a multiplex real-time PCR assay for clarithromycin resistance showed 95% (38/40) concordance with nucleic acid testing and 95% (37/39) concordance with phenotypic testing. This assay provides a simple and rapid alternative to extended incubation or erm(41) sequencing.

  View details for PubMedID 25903572

• Organism burden, toxin concentration, and lactoferrin concentration do not distinguish between clinically significant and nonsignificant diarrhea in patients with Clostridium difficile. Diagnostic microbiology and infectious disease Anikst, V. E., Gaur, R. L., Schroeder, L. F., Banaei, N. 2015

  Abstract

  Clostridium difficile infection is often overdiagnosed in patients with mild diarrhea. We evaluated 4 biomarkers as surrogates for clinically significant diarrhea (?3 episodes in 24hours) in 59 PCR-positive patients with and 59 PCR-positive patients without clinically significant diarrhea. Organism burden (median tcdB cycle threshold value, 26.9 versus 27.1, P=0.25) and toxin A and B concentrations (toxin A, median, 0 versus 0ng/mL, P=0.42; toxin B, median, 0 versus 0ng/mL, P=0.25) were not significantly different between patients with and without clinically significant diarrhea. Fecal lactoferrin concentrations were significantly increased in patients with clinically significant diarrhea (median, 99.0 versus 55.1?g/mL, P=0.05); however, lactoferrin could not sufficiently classify patients into those with and without clinically significant diarrhea. Interventions that limit C. difficile testing to patients with clinically significant diarrhea are needed to improve the positive predictive value of C. difficile diagnostics.

  View details for PubMedID 26778484

• Reproducibility of interferon gamma (IFN-?) release Assays. A systematic review. Annals of the American Thoracic Society Tagmouti, S., Slater, M., Benedetti, A., Kik, S. V., Banaei, N., Cattamanchi, A., Metcalfe, J., Dowdy, D., van Zyl Smit, R., Dendukuri, N., Pai, M., Denkinger, C. 2014; 11 (8): 1267-1276

  Abstract

  Interferon gamma (IFN-?) release assays for latent tuberculosis infection result in a larger-than-expected number of conversions and reversions in occupational screening programs, and reproducibility of test results is a concern.Knowledge of the relative contribution and extent of the individual sources of variability (immunological, preanalytical, or analytical) could help optimize testing protocols.We performed a systematic review of studies published by October 2013 on all potential sources of variability of commercial IFN-? release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB). The included studies assessed test variability under identical conditions and under different conditions (the latter both overall and stratified by individual sources of variability). Linear mixed effects models were used to estimate within-subject SD.We identified a total of 26 articles, including 7 studies analyzing variability under the same conditions, 10 studies analyzing variability with repeat testing over time under different conditions, and 19 studies reporting individual sources of variability. Most data were on QuantiFERON (only three studies on T-SPOT.TB). A considerable number of conversions and reversions were seen around the manufacturer-recommended cut-point. The estimated range of variability of IFN-? response in QuantiFERON under identical conditions was ±0.47 IU/ml (coefficient of variation, 13%) and ±0.26 IU/ml (30%) for individuals with an initial IFN-? response in the borderline range (0.25-0.80 IU/ml). The estimated range of variability in noncontrolled settings was substantially larger (±1.4 IU/ml; 60%). Blood volume inoculated into QuantiFERON tubes and preanalytic delay were identified as key sources of variability.This systematic review shows substantial variability with repeat IFN-? release assays testing even under identical conditions, suggesting that reversions and conversions around the existing cut-point should be interpreted with caution.

  View details for DOI 10.1513/AnnalsATS.201405-188OC

  View details for PubMedID 25188809

• Environmental sampling for Clostridium difficile on alcohol-based hand rub dispensers in an academic medical center. Surgical infections Forrester, J. D., Banaei, N., Buchner, P., Spain, D. A., Staudenmayer, K. L. 2014; 15 (5): 581-584

  Abstract

  Clostridum difficile is a gram-positive, spore-forming anaerobic bacillus that has substantial associated morbidity, mortality, and associated healthcare burdens. Clostridium difficile spores are not destroyed by alcohol. Alcohol gel dispensers are used commonly as the hand sanitization method of choice in hospitals. It is possible that gel dispensers are fomites for C. difficile.Thirty alcohol-based gel dispenser handles outside of rooms of patients with active C. difficile infection were sampled. The samples were assessed for C. difficile by both culture and polymerase chain reaction (PCR). The samples were also assessed for other organisms by culture.No C. difficile was cultured or detected by PCR on any of the gel dispensers. Coagulase-negative Staphyloccus spp., diptheroids, and Bacillus spp. were the organisms detected most commonly.At our institution, C. difficile is not present on alcohol-based gel dispensers, but other potentially pathogenis are.

  View details for DOI 10.1089/sur.2013.102

  View details for PubMedID 25126976

• Environmental Sampling for Clostridium difficile on Alcohol-Based Hand Rub Dispensers in an Academic Medical Center SURGICAL INFECTIONS Forrester, J. D., Banaei, N., Buchner, P., Spain, D. A., Staudenmayer, K. L. 2014; 15 (5): 581-584

  Abstract

  Clostridum difficile is a gram-positive, spore-forming anaerobic bacillus that has substantial associated morbidity, mortality, and associated healthcare burdens. Clostridium difficile spores are not destroyed by alcohol. Alcohol gel dispensers are used commonly as the hand sanitization method of choice in hospitals. It is possible that gel dispensers are fomites for C. difficile.Thirty alcohol-based gel dispenser handles outside of rooms of patients with active C. difficile infection were sampled. The samples were assessed for C. difficile by both culture and polymerase chain reaction (PCR). The samples were also assessed for other organisms by culture.No C. difficile was cultured or detected by PCR on any of the gel dispensers. Coagulase-negative Staphyloccus spp., diptheroids, and Bacillus spp. were the organisms detected most commonly.At our institution, C. difficile is not present on alcohol-based gel dispensers, but other potentially pathogenis are.

  View details for DOI 10.1089/sur.2013.102

  View details for Web of Science ID 000343224800018

• Using cerebrospinal fluid for the diagnosis of tuberculous meningitis with GeneXpert. European respiratory journal Luo, R. F., Gaur, R. L., Banaei, N. 2014; 44 (4): 1094-1095

  View details for DOI 10.1183/09031936.00066214

  View details for PubMedID 25271229

• Mycobacterium tuberculosis Lipoprotein LprG Binds Lipoarabinomannan and Determines Its Cell Envelope Localization to Control Phagolysosomal Fusion PLOS PATHOGENS Shukla, S., Richardson, E. T., Athman, J. J., Shi, L., Wearsch, P. A., McDonald, D., Banaei, N., Boom, W. H., Jackson, M., Harding, C. V. 2014; 10 (10)

  Abstract

  Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (Mtb ?lprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

  View details for DOI 10.1371/journal.ppat.1004471

  View details for Web of Science ID 000344548800055

  View details for PubMedID 25356793

  View details for PubMedCentralID PMC4214796

• LprG-Mediated Surface Expression of Lipoarabinomannan Is Essential for Virulence of Mycobacterium tuberculosis PLOS PATHOGENS Gaur, R. L., Ren, K., Blumenthal, A., Bhamidi, S., Gibbs, S., Jackson, M., Zare, R. N., Ehrt, S., Ernst, J. D., Banaei, N. 2014; 10 (9)

  View details for DOI 10.1371/journal.ppat.1004376

  View details for Web of Science ID 000343014600035

• LprG-mediated surface expression of lipoarabinomannan is essential for virulence of Mycobacterium tuberculosis. PLoS pathogens Gaur, R. L., Ren, K., Blumenthal, A., Bhamidi, S., González-Nilo, F. D., Jackson, M., Zare, R. N., Ehrt, S., Ernst, J. D., Banaei, N. 2014; 10 (9)

  Abstract

  Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.

  View details for DOI 10.1371/journal.ppat.1004376

  View details for PubMedID 25232742

  View details for PubMedCentralID PMC4169494

• Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria. Angewandte Chemie (International ed. in English) Shi, H., Cheng, Y., Lee, K. H., Luo, R. F., Banaei, N., Rao, J. 2014; 53 (31): 8113-8116

  Abstract

  Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant ?-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-?-lactamases in active bacterial pathogens.

  View details for DOI 10.1002/anie.201402012

  View details for PubMedID 24764125

• Multiplex nucleic Acid amplification test for diagnosis of dengue Fever, malaria, and leptospirosis. Journal of clinical microbiology Waggoner, J. J., Abeynayake, J., Balassiano, I., Lefterova, M., Sahoo, M. K., Liu, Y., Vital-Brazil, J. M., Gresh, L., Balmaseda, A., Harris, E., Banaei, N., Pinsky, B. A. 2014; 52 (6): 2011-2018

  Abstract

  Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

  View details for DOI 10.1128/JCM.00341-14

  View details for PubMedID 24671788

• Adult and Pediatric Intra-Institutional Trends of Ciprofloxacin Susceptibility in E. coli Positive Urinary Cultures. Antibiotics (Basel, Switzerland) Owumi, W., Banaei, N., Shortliffe, L. D. 2014; 3 (2): 163-173

  Abstract

  Antimicrobial drug resistance in treatment of urinary tract infection (UTI) continues to rise worldwide. To examine contributions of physician prescribing patterns to fluoroquinolone (ciprofloxacin, CP) resistance, we examined Escherichia coli (E. coli) resistance patterns in urinary cultures. Since CP usage is limited in children, we compared CP resistance trends in adults and children to those of more commonly used trimethoprim-sulfamethoxazole (TMP-SMX) and nitrofurantoin (NF). Our data show that although the general pediatric population has lower resistance to ciprofloxacin, resistance levels are rising with increased usage. While NF susceptibility is historically stable, TMP-SMX resistance is slightly higher in children compared to adults. In both adults and children, antimicrobial resistance patterns vary according to clinical practice site, with ambulatory urology patients showing the highest resistance. This suggests that physician's prescribing patterns contribute to antimicrobial resistance.

  View details for DOI 10.3390/antibiotics3020163

  View details for PubMedID 27025742

• Occupational Screening for Tuberculosis. A Testing Time for Interferon-? Release Assays. Annals of the American Thoracic Society Pai, M., Kik, S. V., Banaei, N. 2014; 11 (3): 399-401

  View details for DOI 10.1513/AnnalsATS.201401-019ED

  View details for PubMedID 24673694

• Economic Evaluation of Laboratory Testing Strategies for Hospital-Associated Clostridium difficile Infection. Journal of clinical microbiology Schroeder, L. F., Robilotti, E., Peterson, L. R., Banaei, N., Dowdy, D. W. 2014; 52 (2): 489-496

  Abstract

  Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.

  View details for DOI 10.1128/JCM.02777-13

  View details for PubMedID 24478478

  View details for PubMedCentralID PMC3911327

• Colorimetric Sensor Array Allows Fast Detection and Simultaneous Identification of Sepsis-Causing Bacteria in Spiked Blood Culture JOURNAL OF CLINICAL MICROBIOLOGY Lim, S. H., Mix, S., Xu, Z., Taba, B., Budvytiene, I., Berliner, A. N., Queralto, N., Churi, Y. S., Huang, R. S., Eiden, M., Martino, R. A., Rhodes, P., Banaei, N. 2014; 52 (2): 592-598

  Abstract

  Sepsis is a medical emergency demanding early diagnosis and tailored antimicrobial therapy. Every hour of delay in initiating effective therapy measurably increases patient mortality. Blood culture is currently the reference standard for detecting bloodstream infection, a multistep process which may take one to several days. Here, we report a novel paradigm for earlier detection and the simultaneous identification of pathogens in spiked blood cultures by means of a metabolomic "fingerprint" of the volatile mixture outgassed by the organisms. The colorimetric sensor array provided significantly faster detection of positive blood cultures than a conventional blood culture system (12.1 h versus 14.9 h, P < 0.001) while allowing for the identification of 18 bacterial species with 91.9% overall accuracy within 2 h of growth detection. The colorimetric sensor array also allowed for discrimination between unrelated strains of methicillin-resistant Staphylococcus aureus, indicating that the metabolomic fingerprint has the potential to track nosocomial transmissions. Altogether, the colorimetric sensor array is a promising tool that offers a new paradigm for diagnosing bloodstream infections.

  View details for DOI 10.1128/JCM.02377-13

  View details for Web of Science ID 000330444200030

  View details for PubMedID 24478493

• Gamma Interferon Release Assays for Detection of Mycobacterium tuberculosis Infection CLINICAL MICROBIOLOGY REVIEWS Pai, M., Denkinger, C. M., Kik, S. V., Rangaka, M. X., Zwerling, A., Oxlade, O., Metcalfe, J. Z., Cattamanchi, A., Dowdy, D. W., Dheda, K., Banaei, N. 2014; 27 (1): 3-20

  Abstract

  Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-?) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers.

  View details for DOI 10.1128/CMR.00034-13

  View details for Web of Science ID 000329324800001

  View details for PubMedID 24396134

• Traveler's encounter with nymphs in a hotel bed. IDCases Sandlund, J., Banaei, N. 2014; 1 (2): 24-25

  Abstract

  This case illustrates skin lesions in a traveler staying in a hotel bed infested with tics. Although infestation of hotels with bedbugs belonging to the Cimex genus is a growing problem worldwide, tick infestation has never been reported before.

  View details for DOI 10.1016/j.idcr.2014.03.002

  View details for PubMedID 26839772

  View details for PubMedCentralID PMC4735455

• Inoculation of QuantiFERON-TB tubes with skin microbiota causes false-positive results. American journal of respiratory and critical care medicine Gaur, R. L., Banaei, N. 2014; 190 (7): 834?37

  View details for PubMedID 25271749

• False-Positive Quantiferon Results at a Large Healthcare Institution. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America Slater, M., Dubose, A., Banaei, N. 2014

  View details for DOI 10.1093/cid/ciu139

  View details for PubMedID 24610428

• Pretreatment of sinus aspirates with dithiothreitol improves yield of fungal cultures in patients with chronic sinusitis INTERNATIONAL FORUM OF ALLERGY & RHINOLOGY Chisholm, K. M., Getsinger, D., Vaughan, W., Hwang, P. H., Banaei, N. 2013; 3 (12): 992-996

  Abstract

  Mold pathogens are a leading cause of chronic rhinosinusitis. Successful isolation of mold on culture is helpful in establishing a diagnosis and guiding therapy. Though mucolytic agents are commonly used in European countries, they are not part of everyday use in North America. In this case-control prospective study, we investigated the yield of fungal culture before and after treatment of sinus aspirates with the mucolytic agent dithiothreitol in a United States hospital.Over a 5-month period during 2011-2012, 359 sinus aspirates from 294 patients with symptoms suspicious for chronic sinusitis or allergic fungal sinusitis were collected. Aspirates were cultured on fungal medium before and after treatment with dithiothreitol.Of the 359 pairs of cultures, 62 (17.3%) demonstrated mold growth on at least 1 of the plates, 9 (14.5%) of which grew more than 1 species of mold. A total of 75 molds were identified, 41 (54.7%) of which were successfully cultured only when the mucus was pretreated with dithiothreitol (p < 0.0001). Quantitatively, more colonies grew from dithiothreitol-treated mucus than from direct-inoculation (p < 0.0001).This study confirms improved recovery of mold from sinus cultures after pretreatment of samples with dithiothreitol. Further studies are needed to correlate these findings with clinical outcome.

  View details for DOI 10.1002/alr.21230

  View details for Web of Science ID 000328300500008

  View details for PubMedID 24124079

• A Pediatric Case of New Delhi Metallo-ß-Lactamase-1-Producing Enterobacteriaceae in The United States. Pediatric infectious disease journal Green, D. A., Srinivas, N., Watz, N., Tenover, F. C., Amieva, M., Banaei, N. 2013; 32 (11): 1291-1294

  Abstract

  We report the second pediatric case of New Delhi metallo-beta-lactamase (NDM-1)-producing Enterobacteriaceae in the United States in a girl from India who presented to a teaching hospital in Northern California with cystitis due to NDM-1-producing E. coli and K. pneumoniae. Laboratory methods included various phenotypic antimicrobial susceptibility testing assays, as well as PCR assays for carbapenemase-encoding genes. Laboratory challenges included a false negative modified Hodge test and reversion of carbapenem resistance in the E. coli strain. The limited number of effective antimicrobial agents and the lack of pediatric-specific safety and efficacy data for these drugs presented significant therapeutic challenges.

  View details for DOI 10.1097/INF.0b013e31829eca34

  View details for PubMedID 23743543

• Impact of Blood Volume, Tube Shaking, and Incubation Time on Reproducibility of QuantiFERON-TB Gold In-Tube Assay. Journal of clinical microbiology Gaur, R. L., Pai, M., Banaei, N. 2013; 51 (11): 3521-3526

  Abstract

  Gamma interferon (IFN-?) release assays (IGRAs) are functional assays used serially to measure the efficacy of novel tuberculosis (TB) vaccines and to screen health care workers for latent tuberculosis infection (LTBI). However, studies have shown nonreproducible IGRA results. In this study, we investigated the effects of blood volume (0.8, 1.0, and 1.2 ml), tube shaking (gentle versus vigorous), and incubation duration (16, 20, and 24 h) on the reproducibility of QuantiFERON-TB Gold In-Tube (QFT-GIT) results for 50 subjects (33 uninfected and 17 infected). The median IFN-? TB response (TB antigen [Ag] minus nil value) was significantly higher with 0.8 ml blood (1.04 IU/ml) than with 1.0 ml (0.85 IU/ml; P = 0.002) or 1.2 ml (0.49 IU/ml; P < 0.001) for subjects with LTBI. Compared with 0.8 ml (11.8%), there were larger proportions of false-negative results with 1.0 ml (29.4%; P = 0.2) and 1.2 ml (41.2%; P = 0.05) of blood for infected subjects. Blood volume did not significantly change the proportions of positive results in uninfected controls. Compared with gentle shaking, vigorous shaking increased the median IFN-? response in nil (0.04 versus 0.06 IU/ml; P < 0.001) and TB Ag (0.12 versus 0.24 IU/ml; P = 0.004) tubes and increased TB responses (TB Agvigorous minus nilgentle) (0.02 versus 0.08 IU/ml; P = 0.004). The duration of incubation did not have a significant impact on the proportion of positive results in uninfected or infected subjects. This study identified blood volume and tube shaking as novel preanalytical sources of variability which require further standardization in order to improve the quality and reproducibility of QFT-GIT results.

  View details for DOI 10.1128/JCM.01627-13

  View details for PubMedID 23966505

• Alerting Physicians during Electronic Order Entry Effectively Reduces Unnecessary Repeat PCR Testing for Clostridium difficile. Journal of clinical microbiology Luo, R. F., Spradley, S., Banaei, N. 2013; 51 (11): 3872-3874

  Abstract

  Hospital information systems (HIS) alerts restricting repeat Clostridium difficile PCR ordering by physicians in patients with a prior result within 7 days eliminated 91% of repeat tests, from 14.5% (282/1,949) repeats preintervention to 1.3% (135/10,285) postintervention. HIS alerting is an effective, targeted, patient-specific tool for improving the quality and utilization of C. difficile results.

  View details for DOI 10.1128/JCM.01724-13

  View details for PubMedID 23985918

• Challenges with QuantiFERON-TB Gold Assay for Large-Scale, Routine Screening of U.S. Healthcare Workers AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE Slater, M. L., Welland, G., Pai, M., Parsonnet, J., Banaei, N. 2013; 188 (8): 1005-1010

  Abstract

  North American occupational health programs that switched from the tuberculin skin test (TST) to IFN-? release assays for latent tuberculosis screening are reporting challenges with interpretation of serial testing results in healthcare workers (HCWs). However, limited data exist on the reproducibility of serial IFN-? release assay results in low-risk HCWs.To evaluate the short-term reproducibility of QuantiFERON-TB Gold In-Tube (QFT) in a large cohort of HCWs and to define a QFT cutoff yielding a conversion rate equivalent to historical TST rates.We retrospectively evaluated the QFT results from HCWs with two or more QFT tests performed between June 2008 and July 2010 at an academic institution. Outcome measures were proportions of reproducibility, quantitative results, and conversion rates with alternate QFT cutoffs.A total of 9,153 HCWs with two or more QFT tests were included in the analysis. Of 8,227 individuals with a negative result, 4.4% (n = 361) converted their QFT result over 2 years. A total of 261 (72.3%) of the HCWs with conversions underwent repeat short-term testing after the first positive result with 64.8% reverting (n = 169). An IFN-? cutoff of 5.3 IU/ml or higher (manufacturer's cutoff is ?0.35 IU/ml) yielded a conversion rate of 0.4%, equal to our institution's historical TST conversion rate.The manufacturer's definition of QFT conversion results in an inflated conversion rate that is incompatible with our low-risk setting. A significantly higher QFT cutoff value is needed to match the historical TST conversion rate. Nonreproducible conversions in most converters suggested false-positive results.

  View details for DOI 10.1164/rccm.201305-0831OC

  View details for Web of Science ID 000325789700021

  View details for PubMedID 23978270

• Occupational screening of health care workers for tuberculosis infection: tuberculin skin testing or interferon-gamma release assays? OCCUPATIONAL MEDICINE-OXFORD Pai, M., Banaei, N. 2013; 63 (7): 458-460

  View details for DOI 10.1093/occmed/kqt105

  View details for Web of Science ID 000327542700002

  View details for PubMedID 24097956

• A Sensitive Multiplex, Real-Time PCR Assay for Prospective Detection of Shiga Toxin-Producing Escherichia coli from Stool Samples Reveals Similar Incidences but Variable Severities of Non-O157 and O157 Infections in Northern California. Journal of clinical microbiology Lefterova, M. I., Slater, K. A., Budvytiene, I., Dadone, P. A., Banaei, N. 2013; 51 (9): 3000-3005

  Abstract

  Rapid and accurate detection of Shiga-toxin producing E. coli of all serotypes from patients with diarrhea is critical for medical management and for prevention of ongoing transmissions. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx1 and stx2 for detection of O157 and non-O157 Shiga-toxin producing E. coli from diarrheal stool samples enriched in GN broth. We show that the assay is 100% sensitive (95% confidence interval (CI), 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%), based on a panel of 40 known STEC-positive and 65 known negative specimens. During a two-year post-validation period, the assay detected a greater number of positive samples from patients in Northern California compared to culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains with 5.8% (1/17) of patients with non-O157 strains and 42.9% (6/14) with O157 strains (P=0.03) developing hemolytic uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to O157 serotype using a sensitive assay. Additionally, a survey of 17 clinical laboratories in Northern California demonstrated that nearly 50% do not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins recommended in the 2009 CDC guidelines.

  View details for DOI 10.1128/JCM.00991-13

  View details for PubMedID 23843484

• Molecular Approaches and Biomarkers for Detection of Mycobacterium tuberculosis. Clinics in laboratory medicine Luo, R. F., Banaei, N. 2013; 33 (3): 553-566

  Abstract

  Tuberculosis (TB) continues to be a public health emergency, compounded by the lack of adequate diagnostic testing in many regions of the world. New advances in the molecular detection of Mycobacterium tuberculosis, including faster and simpler nucleic acid amplification tests, have resulted in rapid and cost-effective methods to diagnose TB and test for drug resistance. Ongoing research on biomarkers for TB infection may lead to new tests for blood, urine, breath, and sputum. Sustained investment in the development and dissemination of diagnostic tests for TB is critical for increasing TB case finding, placing patients on appropriate treatment, and reducing transmission.

  View details for DOI 10.1016/j.cll.2013.03.012

  View details for PubMedID 23931838

• Utility of DNA Sequencing for Direct Identification of Invasive Fungi From Fresh and Formalin-Fixed Specimens AMERICAN JOURNAL OF CLINICAL PATHOLOGY Moncada, P. A., Budvytiene, I., Ho, D. Y., Deresinski, S. C., Montoya, J. G., Banaei, N. 2013; 140 (2): 203-208

  Abstract

  Objectives: To describe and discuss the utility and potential pitfalls of ribosomal RNA locus sequencing for direct identification of invasive fungi from fresh and formalin-fixed, paraffin-embedded specimens. Methods: DNA was extracted from fresh and formalin-fixed, paraffin-embedded tissue and subjected to real-time polymerase chain reaction (PCR) targeting ITS2 and D2 regions of fungal ribosomal RNA locus. Cycle sequencing was performed on PCR products, and the identity of sequences was determined using a public database. Results: Four clinical cases of invasive fungal infection are presented to illustrate the utility of DNA sequencing for determining etiology when microbiological culture is negative, for shortening the time to identification of slow-growing fungi, for guiding antifungal therapy, and for shedding light on the pathogenesis of disseminated fungal infection. Conclusions: Fungal ribosomal RNA locus sequencing from fresh or formalin-fixed, paraffin-embedded specimens is a powerful tool for rapid and accurate diagnosis of patients with culture-negative or uncultured invasive mycosis.

  View details for DOI 10.1309/AJCPNSU2SDZD9WPW

  View details for PubMedID 23897255

• Sorting Inactivated Cells Using Cell-Imprinted Polymer Thin Films ACS NANO Ren, K., Banaei, N., Zare, R. N. 2013; 7 (7): 6031-6036

  Abstract

  Previous work showed that cell imprinting in a polydimethylsiloxane (PDMS) film produced artificial receptors to cells by template-assisted rearrangement of functional groups on the surface of the polymer thin film which facilitated cell capture in the polymer surface indentations by size, shape, and most importantly chemical recognition. We report here that inactivation of cells by treatment with formaldehyde (4%), or glutaraldehyde (2%), or a combination of the two leads to markedly improved capture selectivity (a factor of 3) when cells to be analyzed are inactivated in the same manner. The enhanced capture efficiency compared to living cells results from two factors: (1) rigidification of the cell surface through crosslinking of amine groups by the aldehyde; and (2) elimination of chemicals excreted from living cells which interfere with the fidelity of the cell imprinting process. Moreover, cell inactivation has the advantage of removing biohazard risks associated with working with virulent bacteria. These results are demonstrated using different strains of mycobacterium tuberculosis.

  View details for DOI 10.1021/nn401768s

  View details for Web of Science ID 000322417400045

  View details for PubMedID 23725546

• Images in clinical medicine. Strongyloides stercoralis embryonated ova in the lung. New England journal of medicine Schroeder, L., Banaei, N. 2013; 368 (12)

  View details for DOI 10.1056/NEJMicm1204579

  View details for PubMedID 23514310

• Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak PLOS ONE Kato-Maeda, M., Ho, C., Passarelli, B., Banaei, N., Grinsdale, J., Flores, L., Anderson, J., Murray, M., Rose, G., Kawamura, L. M., Pourmand, N., Tariq, M. A., Gagneux, S., Hopewell, P. C. 2013; 8 (3)

  Abstract

  Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods.To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina's sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case.Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.

  View details for DOI 10.1371/journal.pone.0058235

  View details for Web of Science ID 000315637900110

  View details for PubMedID 23472164

• Trends in incidence and susceptibility among methicillin-resistant Staphylococcus aureus isolated from intranasal cultures associated with rhinosinusitis. American journal of rhinology & allergy Rujanavej, V., Soudry, E., Banaei, N., Baron, E. J., Hwang, P. H., Nayak, J. V. 2013; 27 (2): 134-137

  Abstract

  Reports regarding the incidence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in rhinosinusitis (RS) are limited. This study was designed to identify epidemiology and trends of MRSA incidence and antimicrobial resistance in the sinonasal cavities.This is a retrospective case series. All intranasal/sinus cultures obtained by otolaryngologists at Stanford over a 20-year period (1990-2010) were retrospectively reviewed by mining the microbiology database. Nested searches were then made for all S. aureus and MRSA cultures. Patterns of incidence and changes in antibiotic susceptibilities were tabulated and statistical analysis was performed.Our search retrieved 10,387 positive intranasal culture samples, with S. aureus found in 800 (7.7%), and MRSA comprising 110 (1.06%) of this subset. Between the years of 1990 and 1999, only 2/112 (1.7%) of S. aureus-positive nasal cultures were positive for MRSA, with a sharp rise in incidence to 86/606 (14.2%) from 2000 to 2005, and to 22/82, 26.8% from 2006 to 2010. On a percent basis, using logistic regression modeling, this represents a statistically significant increasing trend (p < 0.0001) for MRSA sinusitis. However, over the 20-year interval studied, the patterns of antibiotic resistance among MRSA remained unaltered, especially with regard to trimethoprim-sulfamethoxazole and vancomycin.S. aureus and MRSA isolates from intranasal cultures, which were essentially absent before the year 2000, became significantly more common earlier this decade. These data show the increased role of MRSA in sinusitis. MRSA antibiotic susceptibilities have remained, however, largely stable during this time period.

  View details for DOI 10.2500/ajra.2013.27.3858

  View details for PubMedID 23562203

• Interferon ?-Release Assays for Diagnosis of Latent Tuberculosis in Healthcare Workers in Low-Incidence Settings: Pros and Cons. Clinical chemistry Pollock, N. R., McAdam, A. J., Pai, M., Nardell, E. A., Bernardo, J., Banaei, N., Mobo, J. 2013

  View details for DOI 10.1373/clinchem.2012.201178

  View details for PubMedID 24100806

• In Vitro Immunomodulation of a Whole Blood IFN-gamma Release Assay Enhances T Cell Responses in Subjects with Latent Tuberculosis Infection PLOS ONE Gaur, R. L., Suhosk, M. M., Banaei, N. 2012; 7 (10)

  Abstract

  Activation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-?) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-? and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

  View details for DOI 10.1371/journal.pone.0048027

  View details for Web of Science ID 000310705300033

  View details for PubMedID 23144722

  View details for PubMedCentralID PMC3483295

• Investigation of False-Positive Results Given by the QuantiFERON-TB Gold In-Tube Assay JOURNAL OF CLINICAL MICROBIOLOGY Slater, M., Parsonnet, J., Banaei, N. 2012; 50 (9): 3105-3107

  Abstract

  We investigated a sudden increase in the rate of positive QuantiFERON-TB Gold In-Tube results from 10% to 31% at a U.S. academic institution. Direct comparison of the TB antigen tubes with tubes from a different lot number identified that a potential problem with the TB antigen vials in a certain tube lot was the likely cause of the elevated positive rate. The underlying defect remains unknown. This finding warrants refinement of quality control programs by the manufacturer and users.

  View details for DOI 10.1128/JCM.00730-12

  View details for Web of Science ID 000307941900046

  View details for PubMedID 22785197

  View details for PubMedCentralID PMC3421800

• Performance of BinaxNOW for Diagnosis of Malaria in a US Hospital JOURNAL OF CLINICAL MICROBIOLOGY DiMaio, M. A., Pereira, I. T., George, T. I., Banaei, N. 2012; 50 (9): 2877-2880

  Abstract

  Microscopic diagnosis and species identification of Plasmodium in areas of nonendemicity provide a robust method for malaria diagnosis but are technically challenging. A prospective study was conducted to measure the performance of BinaxNOW compared to microscopy (the gold standard) in a U.S. teaching hospital. Overall, BinaxNOW was 84.2% sensitive and 99.8% specific. Excluding patients on antimalarial therapy, the sensitivity was 92.9%. Importantly, BinaxNOW initially misclassified a case of Plasmodium falciparum malaria as non-falciparum. These results support the judicious use of BinaxNOW in screening of individuals suspected of having malaria in areas of nonendemicity.

  View details for DOI 10.1128/JCM.01013-12

  View details for PubMedID 22718936

• Sensitivity of QuantiFERON-TB GOLD In-Tube for diagnosis of recent versus remote M.tuberculosis infection DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Bautista, J., Banaei, N. 2012; 73 (3): 257-259

  Abstract

  The sensitivity of QuantiFERON-TB GOLD In-Tube was measured in 104 subjects with recent (?2 years) and remote Mycobacterium tuberculosis infection using tuberculin skin test conversion as the reference standard. The sensitivity was not significantly different between the 2 groups (33% versus 20%, P = 0.3). This finding suggests interferon-? release assays may not be more sensitive for diagnosis of recent than remote infection. Longitudinal studies are needed to validate this finding.

  View details for DOI 10.1016/j.diagmicrobio.2012.03.018

  View details for Web of Science ID 000305546300010

  View details for PubMedID 22521052

• Can a Simple Flotation Method Lower the Limit of Detection of Mycobacterium tuberculosis in Extrapulmonary Samples Analyzed by the GeneXpert MTB/RIF Assay? JOURNAL OF CLINICAL MICROBIOLOGY Taylor, N., Gaur, R. L., Baron, E. J., Banaei, N. 2012; 50 (7): 2272-2276

  Abstract

  The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ? 0.05 and ? 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.

  View details for DOI 10.1128/JCM.01012-12

  View details for Web of Science ID 000307360800017

  View details for PubMedID 22553234

• First Isolation of Cryptococcus uzbekistanensis from an Immunocompromised Patient with Lymphoma JOURNAL OF CLINICAL MICROBIOLOGY Powel, M. S., Alizadeh, A. A., Budvytiene, I., Schaenman, J. M., Banaei, N. 2012; 50 (3): 1125-1127

  Abstract

  Cryptococcus species are known agents of opportunistic infections in healthy and immunocompromised hosts. Here we describe the first case of Cryptococcus uzbekistanensis causing bone marrow infection in an elderly Asian man with undiagnosed T cell lymphoma presenting with fever of unknown origin, pancytopenia, and exposure to chicken manure.

  View details for DOI 10.1128/JCM.05678-11

  View details for Web of Science ID 000300997800099

  View details for PubMedID 22189126

  View details for PubMedCentralID PMC3295126

• IMP-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States JOURNAL OF CLINICAL MICROBIOLOGY Limbago, B. M., Rasheed, J. K., Anderson, K. F., Zhu, W., Kitchel, B., Watz, N., Munro, S., Gans, H., Banaei, N., Kallen, A. J. 2011; 49 (12): 4239-4245

  Abstract

  The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) producing acquired carbapenemases have created a global public health crisis. In the United States, CRE producing the Klebsiella pneumoniae carbapenemase (KPC) are increasingly common and are endemic in some regions. Metallo-?-lactamase (MBL)-producing CRE have recently been reported in the United States among patients who received medical care in countries where such organisms are common. Here, we describe three carbapenem-resistant K. pneumoniae isolates recovered from pediatric patients at a single U.S. health care facility, none of whom had a history of international travel. The isolates were resistant to carbapenems but susceptible to aztreonam, trimethoprim-sulfamethoxazole, and fluoroquinolones. The three isolates were closely related to each other by pulsed-field gel electrophoresis and contained a common plasmid. PCR and sequence analysis confirmed that these isolates produce IMP-4, an MBL carbapenemase not previously published as present among Enterobacteriaceae in the United States.

  View details for DOI 10.1128/JCM.05297-11

  View details for Web of Science ID 000298113400036

  View details for PubMedID 21998425

• Preanalytical Delay Reduces Sensitivity of QuantiFERON-TB Gold In-Tube Assay for Detection of Latent Tuberculosis Infection JOURNAL OF CLINICAL MICROBIOLOGY Doberne, D., Gaur, R. L., Banaei, N. 2011; 49 (8): 3061-3064

  Abstract

  The effects of incubation delays on the accuracy of the QuantiFERON-TB gold in-tube assay (QFT-GIT) were measured. Compared to immediate incubation, 6- and 12-hour delays resulted in positive-to-negative reversion rates of 19% (5/26) and 22% (5/23), respectively. These findings underscore the need for standardizing QFT-GIT preanalytical practices.

  View details for DOI 10.1128/JCM.01136-11

  View details for Web of Science ID 000293221900054

  View details for PubMedID 21697332

• Suboptimal Activation of Antigen-Specific CD4(+) Effector Cells Enables Persistence of M. tuberculosis In Vivo PLOS PATHOGENS Bold, T. D., Banaei, N., Wolf, A. J., Ernst, J. D. 2011; 7 (5)

  Abstract

  Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-? without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-? production. During the early phase of infection, ?10% of P25TCRTh1 cells produced IFN-? in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

  View details for DOI 10.1371/journal.ppat.1002063

  View details for Web of Science ID 000291014000043

  View details for PubMedID 21637811

• Clinical Application and Limitations of Interferon-gamma Release Assays for the Diagnosis of Latent Tuberculosis Infection CLINICAL INFECTIOUS DISEASES Herrera, V., Perry, S., Parsonnet, J., Banaei, N. 2011; 52 (8): 1031-1037

  Abstract

  Interferon-release assays (IGRAs) represent advances in tuberculosis immunology and evolutionary biology. IGRAs were designed to replace tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection because of their logistical advantages and enhanced specificity over TST. Although IGRAs and TST have been useful in epidemiologic studies, they lack the sensitivity and reproducibility normally expected from diagnostic tests in clinical practice. In this review, we present an overview of the current recommendations and knowledge in the field and discuss practical approaches in areas of uncertainty related to discordant IGRA results.

  View details for DOI 10.1093/cid/cir068

  View details for Web of Science ID 000289300100014

  View details for PubMedID 21460320

• Brain Abscess Caused by Phaeoacremonium parasiticum in an Immunocompromised Patient JOURNAL OF CLINICAL MICROBIOLOGY McNeil, C. J., Luo, R. F., Vogel, H., Banaei, N., Ho, D. Y. 2011; 49 (3): 1171-1174

  Abstract

  Phaeoacremonium parasiticum is an environmental fungus usually associated with subcutaneous infections. We report the first documented case of central nervous system involvement with brain abscess formation in a patient with chronic granulomatous disease and review the literature on Phaeoacremonium parasiticum infections.

  View details for DOI 10.1128/JCM.00830-10

  View details for PubMedID 21191052

• Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites CHEMICAL COMMUNICATIONS Koo, C., Wang, S., Gaur, R. L., Samain, F., Banaei, N., Kool, E. T. 2011; 47 (41): 11435-11437

  Abstract

  Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.

  View details for DOI 10.1039/c1cc14871k

  View details for Web of Science ID 000295696300011

  View details for PubMedID 21935547

• Is Repeat PCR Needed for Diagnosis of Clostridium difficile Infection? JOURNAL OF CLINICAL MICROBIOLOGY Luo, R. F., Banaei, N. 2010; 48 (10): 3738-3741

  Abstract

  Patients with diarrhea, defined as loose or watery stool, and two or more Clostridium difficile tcdB PCR tests within 14 days of each other were investigated. Repeat PCR for 293 patients with a prior negative result yielded negative results in 396 (97.5%) of 406 tests. Ten new positives were detected, including one false positive. Repeat PCR within 7 days appears rarely useful, except for patients with evidence of a new infection.

  View details for DOI 10.1128/JCM.00722-10

  View details for Web of Science ID 000282544700042

  View details for PubMedID 20686078

  View details for PubMedCentralID PMC2953130

• Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay JOURNAL OF CLINICAL MICROBIOLOGY Herrera, V., Yeh, E., Murphy, K., Parsonnet, J., Banaei, N. 2010; 48 (8): 2672-2676

  Abstract

  In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37 degrees C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.

  View details for DOI 10.1128/JCM.00482-10

  View details for PubMedID 20519472

• Comparison of real-time polymerase chain reaction and conventional biochemical methods for identification of Mycobacterium chelonae-Mycobacterium abscessus group to the species level DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Guarin, N., Budvytiene, I., Ghafghaichi, L., Banaei, N. 2010; 67 (4): 333-336

  Abstract

  The Mycobacterium chelonae-Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.

  View details for DOI 10.1016/j.diagmicrobio.2010.03.011

  View details for Web of Science ID 000280468700004

  View details for PubMedID 20638600

• Comparison of Single-Copy and Multicopy Real-Time PCR Targets for Detection of Mycobacterium tuberculosis in Paraffin-Embedded Tissue JOURNAL OF CLINICAL MICROBIOLOGY Luo, R. F., Scahill, M. D., Banaei, N. 2010; 48 (7): 2569-2570

  Abstract

  Real-time PCR can rapidly identify Mycobacterium tuberculosis in paraffin-embedded tissue in the absence of microbiological culture. In a comparison of single-copy and multicopy PCR targets in 70 tissue samples, the sensitivities were 26% and 54%, respectively, with 100% specificity. Sensitivity was 75% for newer samples and was not decreased for acid-fast bacillus (AFB) stain-negative specimens.

  View details for DOI 10.1128/JCM.02449-09

  View details for PubMedID 20463168

• Mixed infection involving Actinomyces, Aggregatibacter, and Fusobacterium species presenting as perispinal tumor ANAEROBE Ghafghaichi, L., Troy, S., Budvytiene, I., Banaei, N., Baron, E. J. 2010; 16 (2): 174-178

  Abstract

  A representative case in which a polymicrobial infection involving Fusobacterium nucleatum, Actinomyces israelii and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans was initially diagnosed as malignancy in an edentulous patient. Additional history obtained after the nature of the syndrome was elucidated revealed that he had had his two remaining teeth extracted four months prior to this episode.

  View details for DOI 10.1016/j.anaerobe.2009.07.003

  View details for Web of Science ID 000277734600018

  View details for PubMedID 19628046

• Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci JOURNAL OF CLINICAL MICROBIOLOGY Nguyen, D. T., Yeh, E., Perry, S., Luo, R. F., Pinsky, B. A., Lee, B. P., Sisodiya, D., Baron, E. J., Banaei, N. 2010; 48 (3): 785-790

  Abstract

  Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to beta-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.

  View details for DOI 10.1128/JCM.02150-09

  View details for Web of Science ID 000274996200016

  View details for PubMedID 20071556

  View details for PubMedCentralID PMC2832423

• Preferential Lower Respiratory Tract Infection in Swine-Origin 2009 A(H1N1) Influenza CLINICAL INFECTIOUS DISEASES Yeh, E., Luo, R. F., Dyner, L., Hong, D. K., Banaei, N., Baron, E. J., Pinsky, B. A. 2010; 50 (3): 391-394

  Abstract

  We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.

  View details for DOI 10.1086/649875

  View details for Web of Science ID 000273500300014

  View details for PubMedID 20047483

• First documentation of isoniazid reversion in Mycobacterium tuberculosis INTERNATIONAL JOURNAL OF TUBERCULOSIS AND LUNG DISEASE Richardson, E. T., Lin, S. G., Pinsky, B. A., Desmond, E., Banaei, N. 2009; 13 (11): 1347-1354

  Abstract

  Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure.To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost.Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress.Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG codon 300 which reverted from GGG (glycine, G) to a wild-type codon, TGG (tryptophan, W). The H37Rv DeltakatG::katG W300G mutant was resistant to INH, but also showed a deficiency in coping with oxidative stress.This study confirms that, in the absence of INH pressure, some INH-resistant mutants will revert to a drug-susceptible phenotype. This finding may have broader implications for INH-resistant strains and for the clinically useful lifespan of INH.

  View details for Web of Science ID 000271883400007

  View details for PubMedID 19861005

• Comparison of Real-Time PCR and Conventional Biochemical Methods for Identification of Staphylococcus lugdunensis JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Samson, D., Ghafghaichi, L., Baron, E. J., Banaei, N. 2009; 47 (11): 3472-3477

  Abstract

  Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory.

  View details for DOI 10.1128/JCM.00342-09

  View details for Web of Science ID 000271373000013

  View details for PubMedID 19741081

  View details for PubMedCentralID PMC2772579

• Lipoprotein Processing Is Essential for Resistance of Mycobacterium tuberculosis to Malachite Green ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Banaei, N., Kincaid, E. Z., Lin, S. G., Desmond, E., Jacobs, W. R., Ernst, J. D. 2009; 53 (9): 3799-3802

  Abstract

  Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

  View details for DOI 10.1128/AAC.00647-09

  View details for Web of Science ID 000270014200025

  View details for PubMedID 19596883

• Nontuberculous Mycobacteria Infections in Immunocompromised Patients Single Institution Experience JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY Wei, M. C., Banaei, N., Yakrus, M. A., Stoll, T., Gutierrez, K. M., Agarwal, R. 2009; 31 (8): 556-560

  Abstract

  Disseminated infection due to nontuberculous Mycobacterium (NTM) species is rare in pediatrics. Here we report 6 infections affecting 5 patients at a single institution in an immunocompromised population of pediatric oncology and stem cell transplant recipients. The patients presented within a 1-year period with catheter-associated bacteremia. New pulmonary nodules were noted in 4 of the 5 patients. All of the infections were due to rapidly growing NTM. Patients were successfully treated with removal of the infected catheter and combination antibiotic therapy. There are currently no consensus guidelines for treatment of NTM infections in this population, and a therapeutic approach is presented here.

  View details for PubMedID 19641470

• Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests PLOS ONE Yeh, E., Pinsky, B. A., Banaei, N., Baron, E. J. 2009; 4 (7)

  Abstract

  Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

  View details for DOI 10.1371/journal.pone.0006141

  View details for Web of Science ID 000267806300010

  View details for PubMedID 19578541

  View details for PubMedCentralID PMC2700971

• Bartholin's abscess caused by hypermucoviscous Klebsiella pneumoniae JOURNAL OF MEDICAL MICROBIOLOGY Pinsky, B. A., Baron, E. J., Janda, J. M., Banaei, N. 2009; 58 (5): 671-673

  Abstract

  Klebsiella pneumoniae serogroups displaying the hypermucoviscosity phenotype are associated with a distinct clinical syndrome characterized by liver abscesses, bacteraemia and metastatic lesions. We describe here what we believe to be the first reported case of hypermucoviscous K. pneumoniae causing a superficial Bartholin's abscess in the absence of systemic involvement.

  View details for DOI 10.1099/jmm.0.006734-0

  View details for Web of Science ID 000266018900019

  View details for PubMedID 19369531

• Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex, Real-Time PCR JOURNAL OF CLINICAL MICROBIOLOGY Richardson, E. T., Samson, D., Banaei, N. 2009; 47 (5): 1497-1502

  Abstract

  The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n = 96), MAC (n = 97), MCAG (n = 68), and M. mucogenicum (n = 9) and 95% and 100%, respectively, for MFG (n = 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.

  View details for DOI 10.1128/JCM.01868-08

  View details for Web of Science ID 000265641000032

  View details for PubMedID 19297596

  View details for PubMedCentralID PMC2681835

• Challenges and Pitfalls of Morphologic Identification of Fungal Infections in Histologic and Cytologic Specimens A Ten-Year Retrospective Review at a Single Institution AMERICAN JOURNAL OF CLINICAL PATHOLOGY Sangoi, A. R., Rogers, W. M., Longacre, T. A., Montoya, J. G., Baron, E. J., Banaei, N. 2009; 131 (3): 364-375

  Abstract

  Despite the advantages of providing an early presumptive diagnosis, fungal classification by histopathology can be difficult and may lead to diagnostic error. To assess the accuracy of histologic diagnosis of fungal infections vs culture ("gold standard"), we performed a 10-year retrospective review at our institution. Of the 47 of 338 positive mold and yeast cultures with concurrent surgical pathology evaluation without known history of a fungal infection, 37 (79%) were correctly identified based on morphologic features in histologic and/or cytologic specimens. The 10 discrepant diagnoses (21%) included misidentification of septate and nonseptate hyphal organisms and yeast forms. Errors resulted from morphologic mimics, use of inappropriate terminology, and incomplete knowledge in mycology. The accuracy did not correlate with preceding antifungal therapy (P = .14) or use of special stains (P = .34) and was not operator-dependent. Among 8 discrepancies with clinical follow-up available, 2 potential adverse clinical consequences resulted. While histopathologic identification of fungi in tissue sections and cytologic preparations is prone to error, implementation of a standardized reporting format should improve diagnostic accuracy and prevent adverse outcomes.

  View details for DOI 10.1309/AJCP99OOOZSNISCZ

  View details for PubMedID 19228642

• RP105 Facilitates Macrophage Activation by Mycobacterium tuberculosis Lipoproteins CELL HOST & MICROBE Blumenthal, A., Kobayashi, T., Pierini, L. M., Banaei, N., Ernst, J. D., Miyake, K., Ehrt, S. 2009; 5 (1): 35-46

  Abstract

  RP105, phylogenetically related to Toll-like receptor (TLR)-4, is reported to facilitate B cell activation by the TLR4-agonist lipopolysaccharide (LPS)--but to limit LPS-induced cytokine production by antigen-presenting cells. Here, we show that the role of RP105 extends beyond LPS recognition and that RP105 positively regulates macrophage responses to Mycobacterium tuberculosis (Mtb) lipoproteins. Mtb-infected RP105(-/-) mice exhibited impaired proinflammatory cytokine responses associated with enhanced bacterial burden and increased lung pathology. The Mtb 19 kDa lipoprotein induced release of tumor necrosis factor in a manner dependent on both TLR2 and RP105, and macrophage activation by Mtb lacking mature lipoproteins was not RP105 dependent. Thus, mycobacterial lipoproteins are RP105 agonists. RP105 physically interacted with TLR2, and both RP105 and TLR2 were required for optimal macrophage activation by Mtb. Our data identify RP105 as an accessory molecule for TLR2, forming part of the receptor complex for innate immune recognition of mycobacterial lipoproteins.

  View details for DOI 10.1016/j.chom.2008.12.002

  View details for Web of Science ID 000262885700007

  View details for PubMedID 19154986

• Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level JOURNAL OF CLINICAL MICROBIOLOGY Pinsky, B. A., Banaei, N. 2008; 46 (7): 2241-2246

  Abstract

  The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.

  View details for DOI 10.1128/JCM.00347-08

  View details for Web of Science ID 000258906800016

  View details for PubMedID 18508937

  View details for PubMedCentralID PMC2446918

• Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs JOURNAL OF EXPERIMENTAL MEDICINE Wolf, A. J., Desvignes, L., Linas, B., Banaiee, N., Tamura, T., Takatsu, K., Ernst, J. D. 2008; 205 (1): 105-115

  Abstract

  The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4(+) T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4(+) T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4(+) T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.

  View details for DOI 10.1084/jem.20071367

  View details for Web of Science ID 000252507100012

  View details for PubMedID 18158321

• Evaluation of a semi-automated reporter phage assay for susceptibility testing Myobacterium tuberculosis isolates in South Africa TUBERCULOSIS Banaiee, N., January, V., Barthus, C., Lambrick, M., Roditi, D., Behr, M. A., Jacobs, W. R., Steyn, L. M. 2008; 88 (1): 64-68

  Abstract

  In a prospective study conducted by laboratory technologists in a diagnostic laboratory in Cape Town, South Africa, a semi-automated phage-based antibiotic susceptibility assay was implemented and the performance of the luciferase reporter mycobacteriophage (LRP) system for susceptibility testing of clinical Mycobacterium tuberculosis complex (MTC) isolates against rifampin and isoniazid was evaluated. Two hundred consecutive clinical MGIT cultures of MTC species were included in this study. Antibiotic susceptibility assays were set up manually for the LRP and BACTEC radiometric systems (BACTEC) and read in a plate luminometer and the BACTEC 460 instrument, respectively. Discrepant susceptibility results were resolved by the conventional agar proportion method. Of the 200 secondary cultures prepared for this study, 9 (4.5%) were lost to contamination (LRP 4, BACTEC 1, both 4). All of the remaining 191 cultures underwent susceptibility testing by both methods and the overall agreement between the LRP and BACTEC was 98.4% (rifampin 100%; isoniazid 96.9%). Of the 6 discrepant cultures tested by the agar proportion method, 2 gave results in agreement with the LRP. The sensitivity of the LRP for detection of drug-resistant isolates was 100% for both rifampin (n=9) and isoniazid (n=12). The median turnaround time for susceptibility testing was 2 days with the LRP and 9 days with BACTEC. In conclusion, the semi-automated LRP-based assay offers a rapid and practical approach for accurate susceptibility testing of M. tuberculosis cultures in diagnostic laboratories with limited financial resources, but with competent technologists.

  View details for DOI 10.1016/j.tube.2007.08.006

  View details for Web of Science ID 000252573900008

  View details for PubMedID 17980664

• LspA-independent action of globomycin on Mycobacterium tuberculosis JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY Banaiee, N., Jacobs, W. R., Ernst, J. D. 2007; 60 (2): 414-416

  Abstract

  The objective of this study was to investigate the antimicrobial activity and specificity of globomycin, an inhibitor of lipoprotein signal peptidase II (LspA), against Mycobacterium tuberculosis.The mycobactericidal and mycobacteriostatic activity of globomycin was determined by optical density and cfu plating. The specificity of globomycin was determined by western immunoblotting using anti-MPT83 antibody.Globomycin is mycobactericidal at concentrations>or=40 mg/L. However, at 80 mg/L, the processing of the lipoprotein MPT-83 is unaffected and growth-inhibitory effect of globomycin is unchanged in an lspA null mutant (DeltalspA::lspAmut) lacking the putative drug target.Globomycin kills M. tuberculosis through a mechanism that is independent of LspA.

  View details for DOI 10.1093/jac/dkm223

  View details for Web of Science ID 000248986500031

  View details for PubMedID 17579235

• Genornics and the evolution, pathogenesis, and diagnosis of tuberculosis JOURNAL OF CLINICAL INVESTIGATION Ernst, J. D., Trevejo-Nunez, G., Banaiee, N. 2007; 117 (7): 1738-1745

  Abstract

  Tuberculosis kills nearly 2 million people annually, and current approaches to tuberculosis control are expensive, have limited efficacy, and are vulnerable to being overcome by extensively drug-resistant strains of Mycobacterium tuberculosis. Determination of the genome sequence of M. tuberculosis has revolutionized tuberculosis research, contributed to major advances in the understanding of the evolution and pathogenesis of M. tuberculosis, and facilitated development of new diagnostic tests with increased specificity for tuberculosis. In this review, we describe some of the major progress in tuberculosis research that has resulted from knowledge of the genome sequence and note some of the problems that remain unsolved.

  View details for DOI 10.1172/JCI31810

  View details for Web of Science ID 000247837700002

  View details for PubMedID 17607348

• Regulation of Mycobacterium tuberculosis whiB3 in the mouse lung and macrophages INFECTION AND IMMUNITY Banaiee, N., Jacobs, W. R., Ernst, J. D. 2006; 74 (11): 6449-6457

  Abstract

  Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

  View details for DOI 10.1128/IAI.00190-06

  View details for Web of Science ID 000241600500048

  View details for PubMedID 16923787

• Potent inhibition of macrophage responses to IFN-gamma by live virulent Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2 JOURNAL OF IMMUNOLOGY Banaiee, N., Kincaid, E. Z., Buchwald, U., Jacobs, W. R., Ernst, J. D. 2006; 176 (5): 3019-3027

  Abstract

  Mycobacterium tuberculosis is a highly successful pathogen that can persist and cause disease despite an immune response. One potential mechanism for resisting elimination is by inhibiting the action of IFN-gamma. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-gamma, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN-gamma-responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN-gamma by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-gamma, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN-gamma to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-gamma. M. tuberculosis inhibition of responses to IFN-gamma requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-gamma. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-gamma.

  View details for Web of Science ID 000238768000040

  View details for PubMedID 16493060

• alpha-Defensin expression during myelopoiesis: identification of cis and trans elements that regulate expression of NP-3 in rat promyelocytes JOURNAL OF LEUKOCYTE BIOLOGY Yamamoto, C. M., Banaiee, N., Yount, N. Y., Patel, B., Selsted, M. E. 2004; 75 (2): 332-341

  Abstract

  Alpha-defensins are antimicrobial peptides that contribute to innate-immune functions of neutrophils and intestinal Paneth cells. Transcription of alpha-defensin genes occurs early in neutrophilic myelopoeisis. To examine the mechanisms that regulate alpha-defensin gene expression, we analyzed transcription of rat neutrophil alpha-defensin NP-3 in D4 cells, a subclone of the promyelocytic cell line IPC-81. Northern blot analysis showed that D4 cells express fivefold higher levels of alpha-defensin mRNA than the parental cell line in a manner relatively independent of passage number. Increased levels of steady-state mRNA in D4 cells correlated with markedly elevated peptide levels detected by immunocytochemical staining. To identify the cis-acting DNA elements involved in tissue-specific expression, D4 cells were transfected with luciferase reporter constructs containing NP-3 gene 5'-flanking sequences. Analyses of transfected D4 cells demonstrated that the proximal 87 base pair (bp) sequence contained cis-acting DNA elements necessary for optimal promoter activity. Mutational analyses within the 87-bp region suggested the involvement of the CAAT box and a putative polyoma enhancer-binding protein 2/core-binding factor (PEBP2/CBF) site in defensin gene transcription. Transient transfection analyses using tandem repeats of oligonucleotides containing these sequences demonstrated that proximity of the CAAT box and PEBP2/CBF site was important for defensin promoter activity. Electrophoretic mobility shift assays indicated that PEBP2/CBF or a PEBP2/CBF-related protein was involved in a specific protein-DNA interaction occurring within a DNA fragment containing the CAAT and PEBP2/CBF sequences. These data identify functional trans- and cis-elements that regulate rat defensin gene expression in high defensin-expressing promyelocytic cells.

  View details for DOI 10.1189/jlb.0803384

  View details for Web of Science ID 000188791700022

  View details for PubMedID 14634060

• Rapid identification and susceptibility testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter mycobacteriophages JOURNAL OF MEDICAL MICROBIOLOGY Banaiee, N., Bobadilla-del-Valle, M., Riska, P. F., Bardarov, S., Small, P. M., Ponce-de-Leon, A., Jacobs, W. R., Hatfull, G. F., Sifuentes-Osornio, J. 2003; 52 (7): 557-561

  Abstract

  In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.

  View details for DOI 10.1099/jmm.0.05149-0

  View details for Web of Science ID 000184233400005

  View details for PubMedID 12808076

• Detection and drug-susceptibility testing of M-tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE Bardarov, S., Dou, H., Eisenach, K., Banaiee, N., Ya, S., Chan, J., Jacobs, W. R., Riska, P. F. 2003; 45 (1): 53-61

  Abstract

  Rapid diagnosis of drug-resistant M.tuberculosis (Mtb) is desirable worldwide. We (i) describe a new luciferase reporter phage (LRP), phAE142 for this purpose; (ii) compare it to the automated MGIT 960 for time-to-detection of Mtb in clinical specimens; and (iii) evaluate its use for species confirmation and antibiotic susceptibility testing(AST) of Mtb. Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After "positives" were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST. The LRP method proved comparably efficient to MGIT 960 at detecting Mtb. Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of 3 days, with a sensitivity of 97%. Overall agreement in susceptibility results was 96%. Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16d) with LRP applied to sputum samples.

  View details for Web of Science ID 000181116700007

  View details for PubMedID 12573551

• Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico JOURNAL OF CLINICAL MICROBIOLOGY Banaiee, N., Bodadilla-del-Valle, M., Bardarov, S., Riska, P. F., Small, P. M., Ponce-de-Leon, A., Jacobs, W. R., Hatfull, G. F., Sifuentes-Osornio, J. 2001; 39 (11): 3883-3888

  Abstract

  The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

  View details for Web of Science ID 000171934200011

  View details for PubMedID 11682502

  View details for PubMedCentralID PMC88459

• RAT NEUTROPHIL DEFENSINS - PRECURSOR STRUCTURES AND EXPRESSION DURING NEUTROPHILIC MYELOPOIESIS JOURNAL OF IMMUNOLOGY Yount, N. Y., WANG, M. S., Yuan, J., Banaiee, N., Ouellette, A. J., Selsted, M. E. 1995; 155 (9): 4476-4484

  Abstract

  Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.

  View details for Web of Science ID A1995TB46800045

  View details for PubMedID 7594610