Mandrekar, Jayawant N.

Abstract Background Detection and identification of microorganism(s) involved in periprosthetic joint infection (PJI) can inform surgical management and directed antibiotic therapy. Metagenomic shotgun sequencing is a powerful tool with the potential to change how many PJIs are diagnosed as it allows direct detection and identification of pathogens in clinical specimens. In the largest series to date, we utilized a metagenomics-based approach applied to SF to define potential microbial etiologies of failed total knee arthroplasties (TKAs). Methods Synovial fluid was collected from 112 failed TKAs [74 PJI and 38 aseptic implant failure (AF)] via preoperative arthrocentesis. Cell count and differential, standardized culture and DNA-based metagenomic shotgun sequencing were performed. Human DNA was depleted using the MolYsis basic kit prior to DNA extraction, whole genome amplification, and sequencing. Taxonomic assignment of reads and pathogen identification was achieved using a pipeline incorporating k-mer- and marker gene-based classification software. A scheme for analysis and filtration of false-positives was created and applied, incorporating cut-offs for the number of reads, quality scores, and coverage across a reference genome. Patients were classified as having PJI using the IDSA criteria and expert review. Analyses were recorded as percent agreement, with 95% confidence intervals (CI), of metagenomics to SF culture. Results Metagenomic analysis identified the known pathogen in 54 (90%) (CI, 79.5%–96.2%) of the 60 culture-positive PJIs analyzed and one (2%) (CI, 0.0%–8.9%) potential polymicrobial infection not detected by culture. For the 14 culture-negative PJIs tested, metagenomics showed 79% (CI, 49.2%–95.3%) agreement for negative findings; potential pathogens were identified in three (21%) (CI, 4.7%–50.8%) culture-negative PJI cases, with one being polymicrobial. Of the 37 culture-negative AF cases, metagenomics showed 97% (CI, 85.8%–99.9%) agreement with negative culture and identified one (3%) (CI, 0.0%–14.2%) potential pathogen. For the one culture-positive AF case, metagenomic results were negative, suggesting possible culture contamination. Conclusion Metagenomic shotgun sequencing performed on SF can be used to diagnose PJI and may be particularly useful for culture-negative PJI. Disclosures R. Patel, ASM: Board Member, None; CD Diagnostics, BioFire, Curetis, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, Allergan, and The Medicines Company: Grant Investigator, Grant recipient; Curetis: Consultant, Monies paid to my employer; A patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Patents, Patents, any money is paid to my employer; Actelion: DSMB, Money paid to my employer; ASM and IDSA: Editor’s stipends, Editor’s stipends; NBME, Up-to-Date and the Infectious Diseases Board Review Course: NBME, Up-to-Date and the Infectious Diseases Board Review Course, Honoraria; Roche, ASM, and IDSA: Travel reimbursement, Travel reimbursement

Purpose To begin to quantify and understand the use of the flipped classroom (FC)—a progressive, effective, curricular model—in internal medicine (IM) education in relation to residency program and program director (PD) characteristics. Method The authors conducted a survey that included the Flipped Classroom Perception Instrument (FCPI) in 2015 regarding programs’ use and PDs’ perceptions of the FC model. Results Among the 368 IM residency programs, PDs at 227 (61.7%) responded to the survey and 206 (56.0%) completed the FCPI. Regarding how often programs used the FC model, 34 of the 206 PDs (16.5%) reported “never”; 44 (21.4%) reported “very rarely”; another 44 (21.4%) reported “somewhat rarely”; 59 (28.6%) reported “sometimes”; 16 (7.8%) reported “somewhat often”; and 9 (4.4%) reported “very often.” The mean FCPI score (standard deviation [SD]) for the in-class application factor (4.11 [0.68]) was higher (i.e., more favorable) than for the preclass activity factor (3.94 [0.65]) ( P < .001). FC perceptions (mean [SD]) were higher among younger PDs (≤ 50 years, 4.12 [0.62]; > 50 years, 3.94 [0.61]; P = .04) and women compared with men (4.28 [0.56] vs. 3.91 [0.62]; P < .001). PDs with better perceptions of FCs had higher odds of using FCs (odds ratio, 4.768; P < .001). Conclusions Most IM programs use the FC model at least to some extent, and PDs prefer the interactive in-class components over the independent preclass activities. PDs who are women and younger perceived the model more favorably.

ABSTRACT Eighty Gram-negative bacilli (54 Enterobacteriaceae and 26 nonfermenting Gram-negative bacilli) obtained from multiple institutions in the United States were distributed in a blinded manner to seven testing laboratories to compare their performance of a test for detection of carbapenemase production, the Carba NP test. The Carba NP test was performed by all laboratories, following the Clinical and Laboratory Standards Institute (CLSI) procedure. Site-versus-site comparisons demonstrated a high level of consistency for the Carba NP assay, with just 3/21 site comparisons yielding a difference in sensitivity ( P < 0.05). Previously described limitations with bla OXA-48-like carbapenemases and bla OXA carbapenemases associated with Acinetobacter baumannii were noted. Based on these data, we demonstrate that the Carba NP test, when implemented with the standardized CLSI methodology, provides reproducible results across multiple sites for detection of carbapenemases.

ABSTRACT Hepatitis E virus (HEV) has emerged as a cause of chronic hepatitis among immunocompromised patients. Molecular assays have become important tools for the diagnosis and management of these chronically infected patients. A real-time reverse transcription-quantitative PCR (RT-qPCR) assay utilizing Pleiades probe chemistry and an RNA internal control for the simultaneous detection and quantification of HEV RNA in human serum was developed based on an adaptation of a previously described and broadly reactive primer set targeting the overlapping open reading frame 2/3 (ORF2/3) nucleotide sequence of HEV. A chimeric bovine viral diarrhea virus construct containing an HEV RNA insert (SynTura HEV) was developed, value assigned with the first World Health Organization (WHO) international standard for HEV RNA (code 6329/10), and used to prepare working assay calibrators and controls, which supported an assay quantification range of 100 to 5,000,000 IU/ml. The analytical sensitivity (95% detection rate) of this assay was 25.2 IU/ml (95% confidence interval [CI], 19.2 to 44.1 IU/ml). The assay successfully amplified 16 different HEV sequences with significant nucleotide mismatching in primer/probe binding regions, while evaluation of a WHO international reference panel for HEV genotypes (code 8578/13) showed viral load results falling within the result ranges generated by WHO collaborative study participants for all panel members (genotypes 1 to 4). Broadly reactive RT-qPCR primers targeting HEV ORF2/3 were successfully adapted for use in an assay based on Pleiades probe chemistry. The availability of secondary standards calibrated to the WHO HEV international standard can improve the standardization and performance of assays for the detection and quantification of HEV RNA.

ABSTRACT Failure to eradicate Helicobacter pylori infection is often a result of antimicrobial resistance, which for clarithromycin is typically mediated by specific point mutations in the 23S rRNA gene. The purpose of this study was to define current patterns of antimicrobial susceptibility in H. pylori isolates derived primarily from the United States and to survey them for the presence of point mutations in the 23S rRNA gene and assess the ability of these mutations to predict phenotypic clarithromycin susceptibility. Antimicrobial susceptibility testing was performed using agar dilution on 413 H. pylori isolates submitted to Mayo Medical Laboratories for susceptibility testing. For a subset of these isolates, a 150-bp segment of the 23S rRNA gene was sequenced. A total of 1,970 MICs were reported over the 4-year study period. The rate of clarithromycin resistance was high (70.4%), and elevated MICs were frequently observed for metronidazole (82.4% of isolates had an MIC of >8 μg/ml) and ciprofloxacin (53.5% of isolates had an MIC of >1 μg/ml). A total of 111 archived H. pylori isolates underwent 23S rRNA gene sequencing; we found 95% concordance between genotypes and phenotypes ( P = 0.9802). Resistance to clarithromycin was most commonly due to an A2143G mutation (82%), followed by A2142G (14%) and A2142C (4%) mutations. Clinical H. pylori isolates derived primarily from the United States demonstrated a high rate of clarithromycin resistance and elevated metronidazole and ciprofloxacin MICs. The relative distribution of point mutations at positions 2143 and 2142 in the 23S rRNA gene in clarithromycin-resistant H. pylori was similar to that reported from other parts of the world; these mutations predict phenotypic resistance to clarithromycin.

ABSTRACT Staphylococcal prosthetic joint infections (PJIs) are associated with biofilm formation, making them difficult to treat; if managed with debridement and implant retention, rifampin-based therapy is usually employed. Rifampin resistance potentially challenges PJI treatment. In investigating the effects of rifampin monotherapy on methicillin-resistant Staphylococcus aureus (MRSA) foreign-body osteomyelitis in rats, we previously demonstrated that rifampin resistance was selected but that it disappeared 14 days following rifampin monotherapy (1) and that rifampin resistance occurred less frequently following two rounds than following one round of rifampin monotherapy (2). Here, we compared rifampin monotherapy followed by rifampin-vancomycin combination therapy to rifampin-vancomycin combination therapy alone in experimental MRSA foreign-body osteomyelitis. Animals treated with rifampin monotherapy followed by rifampin-vancomycin combination therapy had decreased quantities of bacteria 14 days following treatment completion ( P = 0.034) compared to those in animals treated with combination therapy alone. Additionally, some isolates recovered from animals treated with combination therapy alone, although still susceptible to rifampin, had higher MIC, minimum biofilm-inhibitory concentration (MBIC), and minimum biofilm-bactericidal concentration (MBBC) values than those of the inoculating strain. This suggests that rifampin may remain a feasible treatment option in foreign-body-associated orthopedic infections following the selection of rifampin resistance.

ABSTRACT We developed a rat model of methicillin-resistant Staphylococcus epidermidis (MRSE) foreign body-associated osteomyelitis and used it to compare tedizolid alone and in combination with rifampin against rifampin alone, vancomycin plus rifampin, and vancomycin alone. A clinical strain of MRSE was inoculated into the proximal tibia, and a stainless steel wire with a precolonized MRSE biofilm was implanted. Following a 1-week infection period, 92 rats received either no treatment ( n = 17) or 14 days of intraperitoneal tedizolid ( n = 15), tedizolid plus rifampin ( n = 15), rifampin ( n = 15), vancomycin plus rifampin ( n = 15), or vancomycin ( n = 15). Quantitative bone and wire cultures were performed after treatment completion and also 1 week after infection in a separate group of five rats. The median quantity of staphylococci in bone after the 1-week infection period was 4.89 log 10 CFU/g bone (interquartile range, 3.83 to 5.33 log 10 CFU/g bone); staphylococci were recovered from all associated wires. A median quantity of staphylococci of 3.70 log 10 CFU/g bone was detected in bones of untreated control rats after 3 weeks. Quantities of staphylococci in bones of all treatment groups except the group receiving vancomycin alone (2.78 log 10 CFU/g) were significantly lower than those for untreated controls, with no staphylococci being detected in the groups receiving rifampin monotherapy, tedizolid-plus-rifampin combination therapy, and vancomycin-plus-rifampin combination therapy. Quantities of staphylococci on wires from all treatment groups that included rifampin were significantly lower than those for untreated controls. No resistance to rifampin, tedizolid, or vancomycin was detected. Tedizolid combined with rifampin was active in a rat model of MRSE foreign body-associated osteomyelitis.

ABSTRACT Foreign-body-associated infections are often difficult to treat, given that the associated microorganisms are in a biofilm state. Previously, we showed that a low-amperage direct electrical current (DC) reduces Propionibacterium acnes biofilms formed on implant-associated materials in vitro . In this study, low-amperage DC was compared to ceftriaxone treatment or no treatment in a novel rat femur model of foreign-body osteomyelitis. A platinum implant seeded with a P. acnes biofilm (10 7 CFU/cm 2 ) and 10 9 CFU of planktonic P. acnes was placed in the femoral medullary cavity. One week later, rats were assigned to one of three treatment groups: no treatment, ceftriaxone treatment, or 200-μA-DC treatment. After 2 weeks of treatment, there were fewer bacteria in the bones of the ceftriaxone group (3.06 log 10 CFU/g of bone [ P = 0.0209]) and the 200-μA-DC group (0.5 log 10 CFU/g [ P = 0.0015]) than in those of the control group (6.58 log 10 CFU/g). The DC-exposed animals exhibited fewer bacteria than the ceftriaxone-treated animals ( P = 0.0330). There were fewer bacteria on the implanted wires in the groups treated with ceftriaxone (0.1 log 10 CFU/cm 2 ) or a 200-μA DC (0.1 log 10 CFU/cm 2 ) than in the control group (2.53 log 10 CFU/cm 2 [ P , 0.0003 for both comparisons]). Low-amperage DC may be useful for treating, or aiding in the treatment of, foreign-body infections caused by P. acnes .