International Spread of Multidrug-Resistant Campylobacter coli in Men Who Have Sex With Men in Washington State and Québec, 2015-2018.

BACKGROUND: Campylobacter species are among the most common causes of enteric bacterial infections worldwide. Men who have sex with men (MSM) are at increased risk for sexually transmitted enteric infections, including globally distributed strains of multidrug-resistant Shigella species. METHODS: This was a retrospective study of MSM-associated Campylobacter in Seattle, Washington and Montréal, Québec with phenotypic antimicrobial resistance profiles and whole genome sequencing (WGS). RESULTS: We report the isolation of 2 clonal lineages of multidrug-resistant Campylobacter coli from MSM in Seattle and Montréal. WGS revealed nearly identical strains obtained from the 2 regions over a 4-year period. Comparison with the National Center for Biotechnology Information's Pathogen Detection database revealed extensive Campylobacter species clusters carrying multiple drug resistance genes that segregated with these isolates. Examination of the genetic basis of antimicrobial resistance revealed multiple macrolide resistance determinants including a novel ribosomal RNA methyltransferase situated in a CRISPR (clustered regularly interspaced short palindromic repeats) array locus in a C. coli isolate. CONCLUSIONS: As previously reported for Shigella, specific multidrug-resistant strains of Campylobacter are circulating by sexual transmission in MSM populations across diverse geographic locations, suggesting a need to incorporate sexual behavior in the investigation of clusters of foodborne pathogens revealed by WGS data.

Comparison of Three Adenovirus Quantitative PCR Assays with ATCC Reference Strains and Clinical Samples.

Adenoviruses (AdV) have been associated with a variety of human diseases and are recognized as causing significant morbidity and mortality in immunocompromised or transplant patients. Quantification of AdV DNA in plasma is notoriously difficult due to the genetic diversity of the 71 different serotypes identified to date. There is no World Health Organization standard available to harmonize quantitative data, so results between labs vary widely. In this study, we compared a laboratory-developed multiplex PCR assay with primers and probes specific for each group (A to G) and subgroup E4 (Octaplex) to one with a single primer and probe set (modified from N. Jothikumar et al., Appl Environ Microbiol 71:3131-3136, 2005) and one utilizing bisulfite pretreatment of DNA to reduce variation prior to amplification (Genetic Signatures). Our Octaplex assay detected all low-copy-number clinical samples, while the other two assays had subsets of samples that did not amplify. The modified Jothikumar assay failed to efficiently amplify three of the high-copy-number cultured strains, while the Genetic Signatures 3base assay had a positive bias, resulting in higher copies/ml (>0.5 log10) for all culture fluids tested. All three assays resulted in endpoint detection of the available 51 AdV types. Using two different materials to generate a standard curve revealed that the Octaplex TaqMan assay and the modified Jothikumar assay both consistently gave adenovirus levels lower than the commercial platform for AdV culture fluids but not patient samples. This study highlights the differences in detection of AdV between laboratories that can be attributed to both the PCR method, as well as the reference material used for quantitation.

Does Size Matter? Comparison of Extraction Yields for Different-Sized DNA Fragments by Seven Different Routine and Four New Circulating Cell-Free Extraction Methods.

An element essential for PCR detection of microbial agents in many sample types is the extraction step, designed to purify nucleic acids. Despite the importance of this step, yields have not been extensively compared across methods to determine whether the method used contributes to quantitative differences and the lack of commutability seen with existing clinical methods. This may in part explain why plasma and blood viral load assays have proven difficult to standardize. Also, studies have identified small DNA fragments of <200 bp in plasma (cell-free DNA [cfDNA]), which may include significant quantities of viral DNA. Our study evaluated extraction yields for 11 commercially available extraction methods, including 4 new methods designed to isolate cfDNA. Solutions of DNA fragments with sizes ranging from 50 to 1,500 bp were extracted, and then the eluates were tested by droplet digital PCR to determine the DNA fragment yield for each method. The results demonstrated a wide range of extraction yields across the variety of methods/instruments used, with the 50- and 100-bp fragment sizes showing especially inconsistent quantitative results and poor yields of less than 20%. Slightly higher, more consistent yields were seen with 2 of the 4 circulating cell-free extraction kits. These results demonstrate a significant need for further evaluation of nucleic acid yields across the variety of extraction platforms and highlight the poor extraction yields of small DNA fragments by existing methods. Further work is necessary to determine the impact of this inconsistency across instruments and the relevance of the low yields for smaller DNA fragments in clinical virology testing.

Kingella kingae Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease.

Kingella kingae is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called csaA that is necessary for synthesis of the [3)-ß-GalpNAc-(1→5)-ß-Kdop-(2→] polysaccharide capsule (type a) in K. kingae strain 269-492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that csaA was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks csaA revealed three additional gene clusters (designated the csb, csc, and csd loci), all encoding predicted glycosyltransferases. The csb locus contains the csbA, csbB, and csbC genes and is associated with a capsule that is a polymer of [6)-α-GlcpNAc-(1→5)-ß-(8-OAc)Kdop-(2→] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-ß-Ribf-(1→2)-ß-Ribf-(1→2)-ß-Ribf-(1→4)-ß-Kdop-(2→] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(O→3)[ß-Galp-(1→4)]-ß-GlcpNAc-(1→3)-α-GlcpNAc-1-] (type d). Introduction of the csa, csb, csc, and csd loci into strain KK01Δcsa, a strain 269-492 derivative that lacks the native csaA gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in K. kingae. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct K. kingae capsule types and suggest that capsule type plays an important role in promoting K. kingae invasive disease.

Genetic and Molecular Basis of Kingella kingae Encapsulation.

Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine (GalNAc) and ß-3-deoxy-d-manno-octulosonic acid (ßKdo). Given the role of capsules as important virulence factors and effective vaccine antigens, we set out to determine the genetic determinants of K. kingae encapsulation. Using a transposon library and a screen for nonencapsulated mutants, we identified the previously identified ctrABCD (ABC transporter) operon, a lipA (kpsC)-like gene, a lipB (kpsS)-like gene, and a putative glycosyltransferase gene designated csaA (capsule synthesis type a gene A). These genes were found to be present at unlinked locations scattered throughout the genome, an atypical genetic arrangement for Gram-negative bacteria that elaborate a capsule dependent on an ABC-type transporter for surface localization. The csaA gene product contains a predicted glycosyltransferase domain with structural homology to GalNAc transferases and a predicted capsule synthesis domain with structural homology to Kdo transferases, raising the possibility that this enzyme is responsible for alternately linking GalNAc to ßKdo and ßKdo to GalNAc. Consistent with this conclusion, mutation of the DXD motif in the GalNAc transferase domain and of the HP motif in the Kdo transferase domain resulted in a loss of encapsulation. Examination of intracellular and surface-associated capsule in deletion mutants and complemented strains further implicated the lipA (kpsC)-like gene, the lipB (kpsS)-like gene, and the csaA gene in K. kingae capsule production. These data define the genetic requirements for encapsulation in K. kingae and demonstrate an atypical organization of capsule synthesis, assembly, and export genes.

The "SEED" Study: The Feasibility of Selecting Patient-Specific Biologically Targeted Therapy with Sorafenib, Everolimus, Erlotinib or Dasatinib for Pediatric and Young Adult Patients with Recurrent or Refractory Brain Tumors.

BACKGROUND: Pediatric brain tumors are the leading cause of cancer death in children and represent a variety of diseases and molecular subtypes. This study sought to evaluate a rapid immunohistochemistry testing panel to aid in therapy selection at the time of malignant tumor recurrence. METHODS: With IRB approval and appropriate informed consent, we conducted a single-institution prospective clinical trial of selected kinase inhibitor therapy. A laboratory-developed immunohistochemical testing panel was performed on tumor tissue, and therapy with one of four small molecule inhibitors was recommended in combination with oral chemotherapy consisting of temozolomide and etoposide. RESULTS: All 20 enrolled subjects were assigned to Everolimus (n = 4), Erlotinib (n = 6) or Dasatinib (n = 10); 90% (18/20) within the pre-specified 14-day feasibility time period. Only two subjects elected treatment on study, 8 received targeted treatment based on testing results either alone (n = 5) or in combination with chemotherapy (n = 3). Other subjects received chemotherapy alone (n = 7), surgery alone (n = 2) or no further therapy (n = 3). Immunohistochemical targets were associated with correlative genetic changes in 28% (5/18) of those evaluated. CONCLUSIONS: It was feasible to rapidly select targeted therapy in recurrent pediatric brain tumors, but not feasible to treat with a uniform combination treatment regimen.

Nursing education challenges: students with English as an additional language.

Nurse educators are challenged by students who did not learn Standard American English as a primary language. It is not only language that makes these students stand out-cultural beliefs, values and practices need to be appreciated as well. The purpose of this article is to synthesize the current qualitative literature on challenges faced in nursing education for students with English as an additional language. Ten qualitative studies regarding educational issues of nursing students with EAL were included in the synthesis. The study was conducted using the ethnographic metasynthesis model of Noblit and Hare. Two major reciprocal translations of educational issues emerged: challenges and reinforcements. Challenges included language, academics, resources, and culture. Reinforcements included resources, academics, and culture. The results may be used by nurse educators for developing interventions to help culturally diverse students succeed. Interventions are directed toward issues surrounding language and culture.

Performance of the Accelerate Diagnostics PhenoTM system with resin-containing BacT/ALERT® Plus blood culture bottles.

Bacteremia and septicemia require rapid identification (ID) and antimicrobial susceptibility testing (AST) to start targeted, appropriate therapy. To answer this need, Accelerate Diagnostics, Inc., developed the Accelerate Pheno™ system (AXDX), a fast ID and phenotypic AST platform. Performance of a pre-FDA clearance version of AXDX was evaluated using 261 positive BacT/ALERT® Plus bottles and compared with standard of care (SOC). Average time to ID was reduced by 24.9±6.9 h and AST by 36.7±18.9 h compared with SOC. AXDX reports ID and AST of blood pathogens in 1.9 and 7.1 h. Positive percent agreement and negative percent agreement of AXDX ID were 94.5% and 98.9%, respectively. AXDX AST had an essential agreement of 96.5% and categorical agreement of 94.6% with 4 major errors and 7 very major errors. AXDX performance was acceptable for all 3 bottle types. Rapid ID and AST with AXDX could impact patient care and antimicrobial stewardship.

Clinical and Genomic Characterization of Recurrent Enterococcal Bloodstream Infection in Patients With Acute Leukemia.

BACKGROUND: Rates and risk factors for recurrent enterococcal bloodstream infection (R-EBSI) and whether the same genetic lineage causes index EBSI and R-EBSI are unknown in patients with acute leukemia (AL) receiving chemotherapy. METHODS: Ninety-two AL patients with EBSI from 2010 to 2015 were included. Enterococcal bloodstream infection was defined by 31 positive blood cultures for Enterococcus faecium or Enterococcus faecalis and fever, hypotension, or chills. Clearance was defined by 31 negative cultures 324 hours after last positive culture and defervescence. Recurrent enterococcal bloodstream infection was defined by a positive blood culture for Enterococcus 324 hours after clearance. Categorical variables were reported as proportions and compared by the χ2 test. Continuous variables were summarized by median and interquartile range (IQR) and compared by the Wilcoxon-Mann-Whitney Test. P values <.05 were considered significant. Whole-genome sequencing was performed on available paired BSI isolates from 7 patients. RESULTS: Twenty-four patients (26%) had 31 episodes of R-EBSI. Median time to R-EBSI (IQR) was 26 (13-50) days. Patients with R-EBSI had significantly longer durations of fever and metronidazole exposure during their index EBSI. Thirty-nine percent of E. faecium R-EBSI isolates became daptomycin-nonsusceptible Enterococcus (DNSE) following daptomycin therapy for index EBSI. Whole-genome sequencing analysis confirmed high probability of genetic relatedness of index EBSI and R-EBSI isolates for 4/7 patients. CONCLUSIONS: Recurrent enterococcal bloodstream infection and DNSE are common in patients with AL and tend to occur within the first 30 days of index EBSI. Duration of fever and metronidazole exposure may be useful in determining risk for R-EBSI. Whole-genome sequencing analysis demonstrates that the same strain causes both EBSI and R-EBSI in some patients.

The Type a and Type b Polysaccharide Capsules Predominate in an International Collection of Invasive Kingella kingae Isolates.

Kingella kingae is an encapsulated Gram-negative bacterium and an important etiology of osteoarticular infections in young children. A recent study examining a diverse collection of carrier and invasive K. kingae isolates from Israel revealed four distinct polysaccharide capsule types. In this study, to obtain a global view of K. kingae capsule type diversity, we examined an international collection of isolates using a multiplex PCR approach. The collection contained all four previously identified capsule types and no new capsule types. Over 95% of invasive isolates in the collection were type a or type b, similar to the findings in Israel. These results suggest that the type a and type b polysaccharide capsules may have enhanced pathogenic properties or may mark clonal groups of strains with specific virulence genes. In addition, they raise the possibility that a vaccine containing the type a and type b capsules might be an effective approach to preventing K. kingae disease. IMPORTANCEKingella kingae has emerged as a significant cause of septic arthritis, osteomyelitis, and bacteremia in young children. A recent study examining a diverse collection of K. kingae isolates from Israel revealed four different polysaccharide capsule types in this species, designated types a to d. To determine the global distribution of K. kingae capsule types, we assembled and capsule typed an international collection of K. kingae isolates. The findings reported here show that the type a and type b capsules represent >95% of the invasive isolates, similar to the Israeli isolate collection, suggesting that a polysaccharide-based vaccine targeting these two capsules could be an attractive approach to prevent K. kingae disease.

Becoming a registered nurse: the nurse extern experience.

Little is known about the perceptions of nursing students externing in newly developed hospital-based programs that focus on socialization and transition to the registered nurse (RN) role rather than on institutional recruitment and retention goals. This qualitative study explored student nurse externs' expectations, experiences, and benefits of participation in a student-focused externship program. Externs wanted to gain experience with skills and learn what it was like to be an RN. Goals were met or exceeded by becoming comfortable in the externship role, growing in skill performance and confidence, and becoming members of the healthcare team. The experience fostered growth from the novice to advanced beginner level of nursing practice. Externs saw the program participation as a valuable way to gain experience and learn what it was like to be an RN.

Characterization of the Kingella kingae polysaccharide capsule and exopolysaccharide.

Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269-492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-ß-GalpNAc-(1→5)-ß-Kdop-(2→ and the other containing galactose alone with the structure →5)-ß-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-ß-GalpNAc-(1→5)-ß-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-ß-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization.