James Stacey Klutts

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infection in immunocompromised patients, which in severe cases leads to fatal meningoencephalitis. Cryptococcus exhibits unique glycobiology that plays important roles in pathogenesis. Unlike model yeast and other common fungal pathogens, Cryptococcus incorporates xylose, a five-carbon monosaccharide, into its glycans. One trimer motif, which consists of xylose in β-1,2 linkage to the reducing mannose of an α-1,3-mannose dimer, occurs in key cryptococcal glycoconjugates that include protein N- and O -linked glycans, glycosylinositol phosphorylceramides (GIPCs), and the capsule polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). We previously identified c ryptococcal β-1,2- x ylosyl t ransferase 1 (Cxt1), which catalyzes formation of this motif in GIPCs, GXM, and GXMGal. Here, we report the discovery of a second enzyme, c ryptococcal β-1,2- x ylosyl t ransferase 2 (Cxt2). Through characterization of cells that lack one or both corresponding genes ( CXT1 and CXT2 ), we have dissected the biological roles of these enzymes, which are overlapping but not identical. Notably, Cxt1 and Cxt2 co-localize in the Golgi, influence capsule in a strain-dependent manner, and together are responsible for all xylose addition to O -glycans. Overall, our work highlights unique roles of these two enzymes and fills a gap in understanding of cryptococcal glycan synthesis. IMPORTANCE: Cryptococcus neoformans is an opportunistic fungal pathogen that causes almost 150,000 deaths each year worldwide. Cryptococcus synthesizes unique glycan structures that play important roles in its biology and pathogenesis. One abundant component of these structures is xylose, a five-carbon monosaccharide. Because xylose is not used by many fungal organisms, including model yeast, we have limited information about how cells add it to their glycans. Here we report a xylosyltransferase enzyme that performs this function, and we characterize specific biological roles of this protein and a closely related one we discovered earlier. We find that these proteins together perform all detectable xylose addition to an important class of protein-linked glycans ( O -glycans). They also both participate in other synthetic processes, although this varies with the specific enzyme and strain background. These results contribute to our understanding of cryptococcal glycan synthesis and underscore the importance of testing multiple background strains.

Control of carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa spread in healthcare settings begins with timely and accurate laboratory testing practices. Survey results show most Veterans Affairs facilities are performing recommended tests to identify these organisms. Most facilities report sufficient resources to perform testing, though medium-complexity facilities report some perceived barriers.

Objective: Develop and implement a system in the Veterans Health Administration (VA) to alert local medical center personnel in real time when an acute- or long-term care patient/resident is admitted to their facility with a history of colonization or infection with a multidrug-resistant organism (MDRO) previously identified at any VA facility across the nation.Methods: An algorithm was developed to extract clinical microbiology and local facility census data from the VA Corporate Data Warehouse initially targeting carbapenem-resistant Enterobacterales (CRE) and methicillin-resistant Staphylococcus aureus (MRSA). The algorithm was validated with chart review of CRE cases from 2010-2018, trialed and refined in 24 VA healthcare systems over two years, expanded to other MDROs and implemented nationwide on 4/2022 as “VA Bug Alert” (VABA). Use through 8/2023 was assessed.Results: VABA performed well for CRE with recall of 96.3%, precision of 99.8%, and F1 score of 98.0%. At the 24 trial sites, feedback was recorded for 1,011 admissions with a history of CRE (130), MRSA (814), or both (67). Among Infection Preventionists and MDRO Prevention Coordinators, 338 (33%) reported being previously unaware of the information, and of these, 271 (80%) reported they would not have otherwise known this information. By fourteen months after nationwide implementation, 113/130 (87%) VA healthcare systems had at least one VABA subscriber.Conclusions: A national system for alerting facilities in real-time of patients admitted with an MDRO history was successfully developed and implemented in VA. Next steps include understanding facilitators and barriers to use and coordination with non-VA facilities nationwide.

Objectives: Blood culture contamination is a major problem in health care, with significant impacts on both patient safety and cost. Initiatives to reduce blood culture contamination require a reliable, consistent metric to track the success of interventions. The objective of our project was to establish a standardized definition of blood culture contamination suitable for use in a Veterans Health Administration (VHA) national data query, then to validate this definition and query. A secondary objective was to construct a national VHA data dashboard to display the data from this query that could be used in VHA quality improvement projects aimed at reducing blood culture contamination. Methods: A VHA microbiology expert work group was formed to generate a standardized definition and oversee the validation studies. The standardized definition was used to generate data for calendar year 2021 using a Structured Query Language data query. Twelve VHA hospital microbiology laboratories compared the data from the query against their own locally derived contamination data and recorded those data in a data collection worksheet that all sites used. Data were collated and presented to the work group. Results: More than 50,000 blood culture accessions were in the validation data set, with more than 1,200 contamination events. The overall blood culture contamination rate for the 12 facilities participating was 2.56% with local definitions and data and 2.43% with the standardized definitions and data query. The main differences noted between the 2 data sets were deemed to be issues in local definitions. The query and definition were then converted into a national data dashboard that all VHA facilities can now access. Conclusions: A standardized definition for blood culture contamination and a national data query were validated for enterprise-wide VHA use. To our knowledge, this represents the first reported standardized, validated, and automated approach for calculating and tracking blood culture contamination. This tool will be key in quality initiatives aimed at reducing contamination events in VHA.

The recent rapid expansion of targeted viral sequencing approaches in conjunction with available bioinformatics have provided an effective platform for studying severe acute respiratory syndrome coronavirus-2 (CoV-2) virions at the molecular level. These means can be adapted to the field of viral molecular epidemiology, wherein localized outbreak clusters can be evaluated and linked. To this end, we have integrated publicly available algorithms in conjunction with targeted RNASeq data in order to qualitatively evaluate similarity or dissimilarity between suspect outbreak strains from hospitals, or assisted living facilities. These tools include phylogenetic clustering and mutational analysis utilizing Nextclade and Ultrafast Sample placement on Existing tRee (UShER). We herein present these outbreak screening tools utilizing three case examples in the context of molecular epidemiology, along with limitations and potential future developments. We anticipate that these methods can be performed in clinical molecular laboratories equipped with CoV-2-sequencing technology.

Laboratory identification of carbapenem-resistant Enterobacteriaceae (CRE) is a key step in controlling its spread. Our survey showed that most Veterans Affairs laboratories follow VA guidelines for initial CRE identification, whereas 55.0% use PCR to confirm carbapenemase production. Most respondents were knowledgeable about CRE guidelines. Barriers included staffing, training, and financial resources.

Introduction: Information continuity is critical to person-centered care when patients receive care from multiple healthcare systems. Patients can access their electronic health record data through patient portals to facilitate information exchange. This pilot was developed to improve care continuity for rural Veterans by (1) promoting the use of the Department of Veterans Affairs (VA) patient portal to share health information with non-VA providers, and (2) evaluating the impact of health information sharing at a community appointment. Materials and Methods: Veterans from nine VA healthcare systems were trained to access and share their VA Continuity of Care Document (CCD) with their non-VA providers. Patients and non-VA providers completed surveys on their experiences. Results: Participants (n = 620) were primarily older, white, and Vietnam era Veterans. After training, 78% reported the CCD would help them be more involved in their healthcare and 86% planned to share it regularly with non-VA providers. Veterans (n = 256) then attended 277 community appointments. Provider responses from these appointments (n = 133) indicated they were confident in the accuracy of the information (97%) and wanted to continue to receive the CCD (96%). Ninety percent of providers reported the CCD improved their ability to have an accurate medication list and helped them make medication treatment decisions. Fifty percent reported they did not order a laboratory test or another procedure because of information available in the CCD. Conclusions: This pilot demonstrates feasibility and value of patient access to a CCD to facilitate information sharing between VA and non-VA providers. Outreach and targeted education are needed to promote consumer-mediated health information exchange.