Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System

Banada, Padmapriya P.; Deshpande, Srinidhi; Chakravorty, Soumitesh; Russo, Riccardo; Occi, James; Meister, Gabriel T.; Jones, Kelly J.; Gelhaus, Carl; Valderas, Michelle Wright; Jones, Martin; Connell, Nancy; Alland, David

doi:10.1128/jcm.01126-16

Cited by 11 publications

(9 citation statements)

References 29 publications

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“…Finally, a rapid time to result may supply actionable information to health care providers during the time of a single patient encounter. In our previous studies, we successfully used the GeneXpert system to detect Mycobacterium tuberculosis (24,25) and Staphylococcus aureus (15) directly from clinical blood samples and Francisella tularensis from the blood of infected macaques (26). The current study demonstrates that a similar approach can detect B. anthracis bacilli in blood at a very low LOD, suggesting that this assay may be clinically useful even if applied at the early stages of a B. anthracis bloodstream infection, since the estimated infective dose can be as low as 10 spores (27).…”

Section: Discussionmentioning

confidence: 99%

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

et al. 2017

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is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range weredetermined by spiking DNA into individual PCR mixtures and CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non- bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the Sterne and V1B strains. There was a 6-log dynamic range. Assay specificity was 100% for tests of non- bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

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Section: Discussionmentioning

confidence: 99%

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

et al. 2017

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“…Molecular beacons were designed using the Mfold web server (20). The F. tularensis assay targeted the multicopy gene ISFtu, and the pag gene was targeted in the B. anthracis assay as described previously (6,9). The Y. pestis assay targeted the pla gene, which encodes a plasminogen activator unique to Y. pestis (21).…”

Section: Methodsmentioning

confidence: 99%

“…Whole-blood sample processing. The GeneXpert system offers the advantage of completely hands-free processing of blood samples, as described previously (6,9,17,23). Similar to these earlier studies, we performed analytical experiments of the TAP multiplex assay by spiking the respective bacteria into deidentified human blood (expired blood from the Rutgers University Hospital [UH] Blood Bank, Newark, NJ) and/or blood collected in K 2 -EDTA tubes obtained from deidentified samples in the UH Hematology Laboratory.…”

Section: Methodsmentioning

confidence: 99%

“…described highly sensitive and rapid assays to detect Staphylococcus aureus (17), F. tularensis (6), and B. anthracis (9) directly from a patient blood sample using the GeneXpert system at cell concentrations at or below 10 CFU/ml. This diagnostic platform uses prefilled assay cartridges that contain all necessary reagents for sample processing, PCR amplification, and target detection.…”

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Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

et al. 2019

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Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis. The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.

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“…A rapid, cartridge-based assay for Francisella tularensis has been developed for use at point of care. 38 A system that uses a sensitive microsphere technology to detect both antibodies and antigens is now available to diagnose infections with Ebola virus and Lassa virus. 39 Although diagnostic ELISA tests are available for anthrax antibodies, 40,41 a compact system (GeneXpert) that includes both sample processing and PCR amplification can produce a result in about 90 minutes.…”

Section: Diagnosis Of Disease Caused By Bioterrorist Agentsmentioning

confidence: 99%

Confronting the threat of bioterrorism: realities, challenges, and defensive strategies

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Cohen

et al. 2019

The Lancet Infectious Diseases

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Global terrorism is a rapidly growing threat to world security, and increases the risk of bioterrorism. In this Review, we discuss the potential threat of bioterrorism, agents that could be exploited, and recent developments in technologies and policy for detecting and controlling epidemics that have been initiated intentionally. The local and international response to infectious disease epidemics, such as the severe acute respiratory syndrome and west African Ebola virus epidemic, revealed serious shortcomings which bioterrorists might exploit when intentionally initiating an epidemic. Development of new vaccines and antimicrobial therapies remains a priority, including the need to expedite clinical trials using new methodologies. Better means to protect health-care workers operating in dangerous environments are also needed, particularly in areas with poor infrastructure. New and improved approaches should be developed for surveillance, early detection, response, effective isolation of patients, control of the movement of potentially infected people, and risk communication. Access to dangerous pathogens should be appropriately regulated, without reducing progress in the development of countermeasures. We conclude that preparedness for intentional outbreaks has the important added value of strengthening preparedness for natural epidemics, and vice versa. This is the second in a Series of three papers about terrorism and health in collaboration with The Lancet Psychiatry

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Sensitive Detection of Francisella tularensis Directly from Whole Blood by Use of the GeneXpert System

Cited by 11 publications

References 29 publications

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System

Multiplex Detection of Three Select Agents Directly from Blood by Use of the GeneXpert System

Confronting the threat of bioterrorism: realities, challenges, and defensive strategies

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