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Article: Multiplex TaqMan

Ross, Anne-Marie L / Stokes, John V / Cross, Claire E / Alugubelly, Navatha / Varela-Stokes, Andrea S

2022 Volume 11, Issue 5

Abstract: Spotted Fever Rickettsiosis (SFR) is caused by spotted fever ... ...

Abstract	Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group
Language	English
Publishing date	2022-05-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens11050594
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis.

Cross, Claire E / Stokes, John V / Alugubelly, Navatha / Ross, Anne-Marie L / Willeford, Bridget V / Walker, Jamie D / Varela-Stokes, Andrea S

Pathogens (Basel, Switzerland)

2022 Volume 11, Issue 2

Abstract: Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the ... ...

Abstract	Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host-pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens,
Language	English
Publishing date	2022-01-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens11020119
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Article: Beyond the IFA: Revisiting the ELISA as a More Sensitive, Objective, and Quantitative Evaluation of Spotted Fever Group

Alugubelly, Navatha / Stokes, John V / Cross, Claire E / Ross, Anne-Marie L / Crawford, Anna E / Fiihr, Gabrielle F / Varela-Stokes, Andrea S

Pathogens (Basel, Switzerland)

2021 Volume 10, Issue 2

Abstract: Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever ... ...

Abstract	Based on limited serological studies, at least 10% of the US population has been exposed to spotted fever group
Language	English
Publishing date	2021-01-20
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens10020088
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Article ; Online: Multiplex TaqMan ® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick- Rickettsia System

Anne-Marie L. Ross / John V. Stokes / Claire E. Cross / Navatha Alugubelly / Andrea S. Varela-Stokes

Pathogens, Vol 11, Iss 594, p

2022 Volume 594

Abstract: Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a ... ...

Abstract	Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig- Amblyomma - Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan ® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork.
Keywords	spotted fever rickettsiosis ; Amblyomma maculatum ; Amblyomma americanum ; Rickettsia parkeri ; Rickettsia amblyommatis ; Cavia porcellus ; Medicine ; R
Subject code	630
Language	English
Publishing date	2022-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: A Multimodal Intervention to Reduce C. difficile Infections and Stool Testing.

Cotter, Jillian M / Stokes, Claire L / Tong, Suhong / Birkholz, Meghan / Child, Jason / Cost, Carrye / Coughlin, Rebecca / Cox, Stephanie / Dolan, Susan A / Dorris, Kathleen / Hazleton, Keith Z / Lugo, Virginia / Norcross, Mikayla / Pearce, Kelly / Dominguez, Samuel R

Pediatrics

2024 Volume 153, Issue 3

Abstract: Background and objectives: The introduction of multiplex gastrointestinal panels at our institution resulted in increased Clostridioides difficile (C. difficile) detection and stool test utilization. We aimed to reduce hospital-onset C. difficile ... ...

Abstract	Background and objectives: The introduction of multiplex gastrointestinal panels at our institution resulted in increased Clostridioides difficile (C. difficile) detection and stool test utilization. We aimed to reduce hospital-onset C. difficile infections (HO-CDIs), C. difficile detection, and overall stool testing by 20% within 1 year. Methods: We conducted a quality improvement project from 2018 to 2020 at a large children's hospital. Interventions included development of a C. difficile testing and treatment clinical care pathway, new options for gastrointestinal panel testing with or without C. difficile (results were suppressed if not ordered), clinical decision support tool to restrict testing, and targeted prevention efforts. Outcomes included the rate of HO-CDI (primary), C. difficile detection, and overall stool testing. All measures were evaluated monthly among hospitalized children per 10 000 patient-days (PDs) using statistical process-control charts. For balancing measures, we tracked suppressed C. difficile results that were released during real-time monitoring because of concern for true infection and C. difficile-related adverse events. Results: HO-CDI decreased by 55%, from 11 to 5 per 10 000 PDs. C. difficile detection decreased by 44%, from 18 to 10 per 10 000 PDs, and overall test utilization decreased by 29%, from 99 to 70 per 10 000 PDs. The decrease in stool tests resulted in annual savings of $55 649. Only 2.3% of initially suppressed positive C. difficile results were released, and no patients had adverse events. Conclusions: Diagnostic stewardship strategies, coupled with an evidence-based clinical care pathway, can be used to decrease C. difficile and improve overall test utilization.
MeSH term(s)	Child ; Humans ; Clostridioides difficile ; Child, Hospitalized ; Clostridium Infections/diagnosis ; Clostridium Infections/prevention & control ; Critical Pathways ; Hospitals, Pediatric
Language	English
Publishing date	2024-02-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	207677-9
ISSN	1098-4275 ; 0031-4005
ISSN (online)	1098-4275
ISSN	0031-4005
DOI	10.1542/peds.2023-061981
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Article ; Online: Evaluating the Clinical and Immune Responses to Spotted Fever Rickettsioses in the Guinea Pig-Tick-Rickettsia System.

Stokes, John V / Levin, Michael L / Cross, Claire E / Ross, Anne-Marie L / Snellgrove, Alyssa N / Willeford, Bridget V / Alugubelly, Navatha / Varela-Stokes, Andrea S

Current protocols

2022 Volume 2, Issue 11, Page(s) e584

Abstract: The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in ...

Abstract	The guinea pig was the original animal model developed for investigating spotted fever rickettsiosis (SFR). This model system has persisted on account of the guinea pig's conduciveness to tick transmission of SFR agents and ability to recapitulate SFR in humans through clinical signs that include fever, unthriftiness, and in some cases the development of an eschar. The guinea pig is the smallest animal model for SFR that allows the collection of multiple blood and skin samples antemortem for longitudinal studies. This unit provides the basic protocols necessary to establish, maintain, and utilize a guinea pig-tick-Rickettsia model for monitoring the course of infection and immune response to an infection by spotted fever group Rickettsia (SFGR) that can be studied at biosafety level 2 (BSL-2) and arthropod containment level 2 (ACL-2); adaptations must be made for BSL-3 agents. The protocols cover methods for tick feeding and colony development, laboratory infection of ticks, tick transmission of Rickettsia to guinea pigs, and monitoring of the course of infection through clinical signs, rickettsial burden, and immune response. It should be feasible to adapt these methods to study other tick-borne pathogens. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tick transmission of SFGR to guinea pigs Support Protocol 1: Laboratory infection of ticks by injection Alternate Protocol 1: Needle inoculation of SFGR to guinea pigs Basic Protocol 2: Monitoring the course of guinea pig rickettsial infection: clinical signs Basic Protocol 3: Monitoring the course of guinea pig rickettsial infection: collection of biological specimens Support Protocol 2: Guinea pig anesthesia Basic Protocol 4: Monitoring rickettsial burden in guinea pigs by multiplex qPCR Basic Protocol 5: Monitoring guinea pig immune response to infection: blood leukocytes by flow cytometry Basic Protocol 6: Monitoring immune response to guinea pig rickettsial infection: leukocyte infiltration of skin at the tick bite site by flow cytometry Basic Protocol 7: Monitoring the immune response to guinea pig rickettsial infection: antibody titer by ELISA Support Protocol 4: Coating ELISA Plates Alternate Protocol 2: Monitoring immune response to guinea pig rickettsial infection: antibody titer by immunofluorescence assay.
MeSH term(s)	Animals ; Guinea Pigs ; Humans ; Disease Models, Animal ; Immunity ; Laboratory Infection ; Rickettsia/physiology ; Spotted Fever Group Rickettsiosis/diagnosis ; Spotted Fever Group Rickettsiosis/immunology ; Ticks/microbiology
Language	English
Publishing date	2022-11-16
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.584
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Article ; Online: Insights Into the Differential Desensitization of

Papke, Roger L / Stokes, Clare

Molecular pharmacology

2022 Volume 103, Issue 2, Page(s) 63–76

Abstract: The development of highly efficacious positive allosteric modulators (PAMs) ... ...

Abstract	The development of highly efficacious positive allosteric modulators (PAMs) of
MeSH term(s)	Ligands ; Receptors, Nicotinic/genetics ; Receptors, Nicotinic/metabolism ; alpha7 Nicotinic Acetylcholine Receptor/genetics ; alpha7 Nicotinic Acetylcholine Receptor/metabolism ; Protein Isoforms ; Allosteric Regulation
Chemical Substances	nicotinic receptor alpha4beta2 ; Ligands ; Receptors, Nicotinic ; alpha7 Nicotinic Acetylcholine Receptor ; Protein Isoforms
Language	English
Publishing date	2022-11-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	124034-1
ISSN	1521-0111 ; 0026-895X
ISSN (online)	1521-0111
ISSN	0026-895X
DOI	10.1124/molpharm.122.000591
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Article: Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis

Cross, Claire E. / Stokes, John V. / Alugubelly, Navatha / Ross, Anne-Marie L. / Willeford, Bridget V. / Walker, Jamie D. / Varela-Stokes, Andrea S.

Pathogens. 2022 Jan. 20, v. 11, no. 2

2022

Abstract	Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host–pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.
Keywords	Mycobacterium ulcerans ; Staphylococcus aureus ; animal models ; antibodies ; dissociation ; flow cytometry ; fluorescence ; guinea pigs ; humans ; immunophenotyping ; inflammation ; leukocytes ; pathogens ; rickettsial diseases ; ticks ; virulence
Language	English
Dates of publication	2022-0120
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens11020119
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Skin in the Game

Claire E. Cross / John V. Stokes / Navatha Alugubelly / Anne-Marie L. Ross / Bridget V. Willeford / Jamie D. Walker / Andrea S. Varela-Stokes

Pathogens, Vol 11, Iss 119, p

An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis

2022 Volume 119

Abstract	Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host–pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans .
Keywords	Cavia porcellus ; tick bite ; tissue dissociation ; innate immunity ; strain 2 ; skin biopsy ; Medicine ; R
Subject code	571
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
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Article ; Online: Severe COVID-19 disease in two pediatric oncology patients.

Stokes, Claire L / Patel, Pratik A / Sabnis, Himalee S / Mitchell, Sarah G / Yildirim, Inci B / Pauly, Melinda G

Pediatric blood & cancer

2020 Volume 67, Issue 9, Page(s) e28432

MeSH term(s)	Adolescent ; Betacoronavirus ; COVID-19 ; Coronavirus Infections/complications ; Coronavirus Infections/diagnosis ; Female ; Hodgkin Disease/complications ; Humans ; Orbital Neoplasms/complications ; Pandemics ; Pneumonia, Viral/complications ; Pneumonia, Viral/diagnosis ; Rhabdomyosarcoma/complications ; SARS-CoV-2
Keywords	covid19
Language	English
Publishing date	2020-07-06
Publishing country	United States
Document type	Case Reports ; Letter
ZDB-ID	2131448-2
ISSN	1545-5017 ; 1545-5009
ISSN (online)	1545-5017
ISSN	1545-5009
DOI	10.1002/pbc.28432
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