Multicenter evaluation of the Selux Next-Generation Phenotyping antimicrobial susceptibility testing system.

The Selux Next-Generation Phenotyping (NGP) system (Charlestown, MA) is a new antimicrobial susceptibility testing system that utilizes two sequential assays performed on all wells of doubling dilution series to determine MICs. A multicenter evaluation of the performance of the Selux NGP system compared with reference broth microdilution was conducted following FDA recommendations and using FDA-defined breakpoints. A total of 2,488 clinical and challenge isolates were included; gram-negative isolates were tested against 24 antimicrobials, and gram-positive isolates were tested against 15 antimicrobials. Data is provided for all organism-antimicrobial combinations evaluated, including those that did and did not meet FDA performance requirements. Overall very major error and major error rates were less than 1% (31/3,805 and 107/15,606, respectively), essential agreement and categorical agreement were >95%, reproducibility was ≥95%, and the average time-to-result (from time of assay start to time of MIC result) was 5.65 hours.

Disseminated Protothecosis Due to Prototheca zopfii and Literature Review.

ABSTRACT: Prototheca species are achlorophyllic algae that are a rare cause of infection in humans. It most commonly causes localized cutaneous disease and rarely disseminated infection. Immunocompromised patients have the highest risk of disseminated protothecosis, with a higher mortality rate than localized cutaneous infections. At the species level, infections caused by Prototheca zopfii are reported less frequently than those caused by Prototheca wickerhamii. The diagnosis can be made using histopathology, culture, and molecular testing. There is no definitive evidence for an effective treatment, which currently consists of antifungals (primarily amphotericin B). With only a handful of cases of disseminated protothecosis reported worldwide that are caused by P. zopfii , we herein present an additional case of a postbone marrow transplant patient in the Midwest of the United States.

Clinical Manifestations and Stool Load of Atypical Enteropathogenic Escherichia coli Infections in United States Children and Adults.

BACKGROUND & AIMS: There is debate whether atypical enteropathogenic Escherichia coli (aEPEC) causes disease in adults. aEPEC is commonly detected in symptomatic and asymptomatic individuals. aEPEC, in contrast to typical EPEC, lacks bundle-forming pili, altering its pathogenicity. Here, we define for the first time the clinical manifestations of sporadic aEPEC infection in United States children and adults and determine whether EPEC load correlates with disease. METHODS: This is a retrospective case-control study of 380 inpatients/outpatients of all ages. EPEC load in stools was determined by quantitative polymerase chain reaction. RESULTS: Diarrhea, vomiting, abdominal pain, and fever were more prevalent in EPEC-positive cases than in EPEC-negative controls. aEPEC infection caused mostly acute, mild diarrhea lasting for 6 to 13 days. However, some had severe diarrhea with 10 to 40 bowel movements per day or had persistent/chronic diarrhea. Fever, vomiting, and abnormal serum sodium levels were more common in children. Adults more often reported abdominal pain and longer duration of diarrhea. Symptomatic aEPEC infection was associated with leukocytosis in 24% of patients. EPEC load >0.1% was associated with symptomatic infection; however, loads varied greatly. Co-infecting pathogens did not alter diarrhea severity or EPEC load. Longitudinal data reveal that some are colonized for months to years or are repeatedly infected. CONCLUSIONS: aEPEC is associated with a wide array of symptoms in adults, ranging from asymptomatic carriage to severe diarrhea. Higher EPEC loads are associated with presence of symptoms, but bacterial load does not predict disease or severity. Future studies are needed to understand bacterial and host factors that contribute to aEPEC pathogenicity to improve diagnostic tools and clinical care.

Recognition of Diagnostic Gaps for Laboratory Diagnosis of Fungal Diseases: Expert Opinion from the Fungal Diagnostics Laboratories Consortium (FDLC).

Fungal infections are a rising threat to our immunocompromised patient population, as well as other nonimmunocompromised patients with various medical conditions. However, little progress has been made in the past decade to improve fungal diagnostics. To jointly address this diagnostic challenge, the Fungal Diagnostics Laboratory Consortium (FDLC) was recently created. The FDLC consists of 26 laboratories from the United States and Canada that routinely provide fungal diagnostic services for patient care. A survey of fungal diagnostic capacity among the 26 members of the FDLC was recently completed, identifying the following diagnostic gaps: lack of molecular detection of mucormycosis; lack of an optimal diagnostic algorithm incorporating fungal biomarkers and molecular tools for early and accurate diagnosis of Pneumocystis pneumonia, aspergillosis, candidemia, and endemic mycoses; lack of a standardized molecular approach to identify fungal pathogens directly in formalin-fixed paraffin-embedded tissues; lack of robust databases to enhance mold identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; suboptimal diagnostic approaches for mold blood cultures, tissue culture processing for Mucorales, and fungal respiratory cultures for cystic fibrosis patients; inadequate capacity for fungal point-of-care testing to detect and identify new, emerging or underrecognized, rare, or uncommon fungal pathogens; and performance of antifungal susceptibility testing. In this commentary, the FDLC delineates the most pressing unmet diagnostic needs and provides expert opinion on how to fulfill them. Most importantly, the FDLC provides a robust laboratory network to tackle these diagnostic gaps and ultimately to improve and enhance the clinical laboratory's capability to rapidly and accurately diagnose fungal infections.

In Vitro Pharmacodynamic Analyses Help Guide the Treatment of Multidrug-Resistant Enterococcus faecium and Carbapenem-Resistant Enterobacter cloacae Bacteremia in a Liver Transplant Patient.

BACKGROUND: Infections due to multidrug-resistant pathogens are particularly deadly and difficult to treat in immunocompromised patients, where few data exist to guide optimal antimicrobial therapy. In the absence of adequate clinical data, in vitro pharmacokinetic (PK)/pharmacodynamic (PD) analyses can help to design treatment regimens that are bactericidal and may be clinically effective. METHODS: We report a case in which in vitro pharmacodynamic analyses were utilized to guide the treatment of complex, recurrent bacteremias due to vancomycin-, daptomycin-, and linezolid-resistant Enterococcus faecium and carbapenem-resistant Enterobacter cloacae complex in a liver transplant patient. RESULTS: Whole-genome sequencing revealed unique underlying resistance mechanisms and explained the rapid evolution of phenotypic resistance and complicated intrahost genomic dynamics observed in vivo. Performing this comprehensive genotypic and phenotypic testing and time-kill analyses, along with knowledge of institution and patient-specific factors, allowed us to use precision medicine to design a treatment regimen that maximized PK/PD. CONCLUSIONS: This work provides a motivating example of clinicians and scientists uniting to optimize care in the era of escalating antimicrobial resistance.

Efficacy of oritavancin alone and in combination against vancomycin-susceptible and -resistant enterococci in an in-vivo Galleria mellonella survival model.

OBJECTIVE: The optimal therapy for serious enterococcal infections, especially vancomycin-resistant enterococci (VRE), remains unclear, although combination therapy is often recommended. Oritavancin has demonstrated in-vitro activity against VRE, but data evaluating oritavancin in combination with other agents and in in-vivo systems are lacking. The objective of this study was to evaluate the efficacy of oritavancin alone and in combination with ceftriaxone, daptomycin, gentamicin, linezolid and rifampin against vancomycin-susceptible enterococci and VRE in an in-vivo Galleria mellonella survival model. METHODS: Five enterococcal strains were used: three clinical isolates (VRE S38141, VRE H19570, VRE W21579), Enterococcus faecium ATCC 700221 and Enterococcus faecalis ATCC 29212. G. mellonella larvae were inoculated with the test strain followed by the test drug at humanized weight-based dose alone or in combination within 1 h of inoculation. After injection, larvae were incubated at 37°C and survival was measured daily for 7 days. Survival was plotted using the Kaplan-Meier method, and differences between groups were determined via the log-rank test. Mean survival times were also determined. RESULTS: Each single agent improved survival significantly compared with the untreated control strain. Oritavancin was the most efficacious single agent, and led to a significant increase in survival compared with ceftriaxone, gentamicin and daptomycin. Compared with oritavancin alone, none of the oritavancin combinations tested were significantly better, and mean survival times were comparable. CONCLUSIONS: Oritavancin monotherapy had the highest survival rate at 7 days, and none of the combinations tested showed improved survival over oritavancin alone. These data add to the body of literature rebutting the routine use of combination therapy with oritavancin for the treatment of infections due to VRE.

Recovery of Cryptococcus gattii from an Infected Ventriculo-Peritoneal Shunt, Illinois, USA.

Cryptococcus gattii is a fungal pathogen endemic in tropical and subtropical regions. Isolated cases and outbreaks have been reported in areas of North America and Europe, expanding the distribution pattern beyond warmer regions. We describe a case of ventriculo-peritoneal shunt infection by C. gattii in an immunocompetent person in Illinois.

Clinical evaluation of Roche COBAS® AmpliPrep/COBAS® TaqMan® CMV test using nonplasma samples.

Cytomegalovirus (CMV) infection is a leading cause of loss of hearing, vision, and mental retardation in congenitally infected children. It is also associated with complications of organ transplant and opportunistic HIV coinfection. The Roche COBAS® AmpliPrep/COBAS® TaqMan® CMV test is an FDA-approved test that measures CMV DNA viral load in plasma for the diagnosis and management of patients at risk of CMV-associated diseases. Besides plasma, CMV is often found in bronchoalveolar lavage (BAL), cerebrospinal fluid (CSF), and urine. Thus, monitoring of CMV for critical care of patients in these nonplasma samples becomes necessary. The objective of this study was to conduct an analytic and clinical feasibility study of the Roche CMV test in BAL, CSF, and urine. The lower limit of detection, analytic measurement range, assay sensitivity, specificity, and precision were determined. Results of this study showed that the lower limit of detections were 50, 100, and 300 IU/mL for BAL, CSF, or urine, respectively. The analytic measurement ranges were from log10 2.48 to log10 5.48. The assay specificity was 94.4% for BAL and 100% for CSF and urine. The assay precision was all within the acceptable range. The performance of Roche test was further compared with 2 comparators including the RealTime CMV assay (Abbott Molecular) and a CMV Quantitative Polymerase Chain Reaction test (Vela Diagnostics). There was a general positive correlation between the Roche method and the Abbott or the Vela method. Overall, this study suggests that the Roche CMV test is suitable for the quantification of CMV viral load DNA in the described nonplasma samples.

Impact of Clinical and Laboratory Standards Institute breakpoint changes on susceptibility rates of cephalosporins in uncomplicated urinary tract infections caused by Enterobacteriaceae.

Breakpoint changes may impact cephalosporin susceptibility rates in uncomplicated urinary tract infections (uUTIs). Applying the ≤16-mg/L breakpoint to urine cultures from adult women in an academic health system resulted in cefazolin being the most active uUTI antimicrobial, with 86.9% susceptibility, compared to levofloxacin (80%), nitrofurantoin (76.5%), and sulfamethoxazole-trimethoprim (72.6%).

Multicenter Evaluation of the Accelerate PhenoTest BC Kit for Rapid Identification and Phenotypic Antimicrobial Susceptibility Testing Using Morphokinetic Cellular Analysis.

We describe results from a multicenter study evaluating the Accelerate Pheno system, a first of its kind diagnostic system that rapidly identifies common bloodstream pathogens from positive blood cultures within 90 min and determines bacterial phenotypic antimicrobial susceptibility testing (AST) results within ∼7 h. A combination of fresh clinical and seeded blood cultures were tested, and results from the Accelerate Pheno system were compared to Vitek 2 results for identification (ID) and broth microdilution or disk diffusion for AST. The Accelerate Pheno system accurately identified 14 common bacterial pathogens and two Candida spp. with sensitivities ranging from 94.6 to 100%. Of fresh positive blood cultures, 89% received a monomicrobial call with a positive predictive value of 97.3%. Six common Gram-positive cocci were evaluated for ID. Five were tested against eight antibiotics, two resistance phenotypes (methicillin-resistant Staphylococcus aureus and Staphylococcus spp. [MRSA/MRS]), and inducible clindamycin resistance (MLSb). From the 4,142 AST results, the overall essential agreement (EA) and categorical agreement (CA) were 97.6% and 97.9%, respectively. Overall very major error (VME), major error (ME), and minor error (mE) rates were 1.0%, 0.7%, and 1.3%, respectively. Eight species of Gram-negative rods were evaluated against 15 antibiotics. From the 6,331 AST results, overall EA and CA were 95.4% and 94.3%, respectively. Overall VME, ME, and mE rates were 0.5%, 0.9%, and 4.8%, respectively. The Accelerate Pheno system has the unique ability to identify and provide phenotypic MIC and categorical AST results in a few hours directly from positive blood culture bottles and support accurate antimicrobial adjustment.

Deep brain stimulator infection by a novel rapid growing mycobacterium.

Devise-related infections after deep brain stimulator implantation are not uncommon. However, infections due to mycobacteria have not been reported in the medical literature. We describe the first reported case of DBS infection due to a novel rapidly growing mycobacteria, most closely resembling Mycobacterium goodii, by rpoB gene sequencing.

Synergistic activity of ceftazidime-avibactam and aztreonam against serine and metallo-ß-lactamase-producing gram-negative pathogens.

This study assessed the in vitro synergy between ceftazidime, aztreonam, and ceftazidime-avibactam against serine and metallo-ß-lactamase (MBL)-producing pathogens via the Etest MIC:MIC ratio and Agar-Etest synergy methods. The combination of aztreonam and ceftazidime-avibactam was synergistic against all Enterobacteriaceae. None of the tested combinations were consistently synergistic against IMP-producing P. aeruginosa.

Pasteurella multocida urinary tract infection in a patient with cervical cancer.

Introduction. Infections caused by Pasteurella species are commonly associated with contact with dogs and cats, typically involving bites and scratches, but casual contact with household pets can also be a risk factor. Urinary tract infection (UTI) caused by Pasteurella species is rare and a significant majority of cases have some known risk factor associated with an underlying chronic illness or structural and/or functional urological abnormality. Case presentation. Here, we present a case of a UTI due to Pasteurella multocida in a patient with squamous cell carcinoma of the cervix who also had a household cat. Conclusion. Providers and laboratorians should be aware of risk factors associated with UTIs caused by Pasteurella species.

In Vitro Activity of Retapamulin and Antimicrobial Susceptibility Patterns in a Longitudinal Collection of Methicillin-Resistant Staphylococcus aureus Isolates from a Veterans Affairs Medical Center.

Mupirocin is a topical antimicrobial used to decolonize patients who carry methicillin-resistant Staphylococcus aureus (MRSA), and the topical agent retapamulin may be a potential alternative therapy. The goal of this study was to determine the in vitro activity of retapamulin as well as a panel of 15 antimicrobial agents, including mupirocin, for 403 MRSA isolates collected longitudinally from a naive population at the Veterans Affairs Puget Sound Health Care System. The MICs for retapamulin had a unimodal distribution, ranging from 0.008 to 0.5 µg/ml. One isolate had an MIC of >16 µg/ml, was also resistant to clindamycin and erythromycin, and was recovered from the nares of a patient undergoing hemodialysis. Twenty-four isolates (6%) and 11 isolates (3%) demonstrated low-level resistance (MICs of 8 to 64 µg/ml) and high-level resistance (MICs of ≥ 512 µg/ml), respectively, to mupirocin. Isolates were recovered from 10 patients both before and after mupirocin therapy. Of those, isolates from 2 patients demonstrated MIC changes postmupirocin therapy; in both cases, however, strain typing demonstrated that the pre- and postmupirocin strains were different. A total of 386 isolates (96%) had vancomycin MICs of ≤ 1.0 µg/ml; 340 isolates (84%) were resistant to levofloxacin, 18 isolates (4.5%) were resistant to trimethoprim-sulfamethoxazole, and 135 isolates (33%) had elevated MICs of 4 µg/ml for linezolid. The baseline levels of resistance were low for mupirocin (9%) and even lower for retapamulin (0.25%) Although the use of mupirocin is currently the standard therapy for decolonization practices, the activity of retapamulin warrants its consideration as an alternative therapy in MRSA decolonization regimens.

Impact of identification of Streptococcus dysgalactiae subspecies equisimilis from throat cultures in an adult population.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) are isolated from the throat of patients with pharyngitis, although the clinical significance remains debated. We sought to determine the incidence and association with pharyngitis of SDSE in an adult veteran population. Organisms were phenotypically identified to subspecies and Lancefield group, with selective 16S rRNA gene sequencing. From 833 throat cultures, the overall frequency of SDSE was 3.4% (64% group C and 36% group G) as compared to 8.6% for S. pyogenes (GAS). SDSE was described as a large colony in only 29% of the original culture evaluations by bench technologists, and clinical symptoms were similar for GAS and SDSE. Laboratory algorithms that are limited to identification of only GAS or are based on Lancefield group or visual identification of "large-colony type" ß hemolytic Lancefield group C and G streptococci may be missing or misidentifying SDSE along with Anginosus group streptococci.

Impact of strain typing methods on assessment of relationship between paired nares and wound isolates of methicillin-resistant Staphylococcus aureus.

The anterior nares are the site of choice for the Veterans Administration methicillin-resistant Staphylococcus aureus (MRSA) surveillance program; however, a correlation between nares colonization and concomitant wound infections has not been well established. The purpose of this study was 3-fold: to determine the relatedness of MRSA isolates from 40 paired wound and nares specimens by four different strain typing methods, to determine concordance of typing methods, and to establish a baseline of MRSA types at this medical center. Isolates were typed by repetitive PCR (rep-PCR) (DiversiLab System; DL) and SpectraCell Raman analysis (SCRA) (commercially available methods that can be performed within a clinical lab), pulsed-field gel electrophoresis (PFGE), and an antibiotic susceptibility profile (AB). Whole-genome optical mapping (WGM) (OpGen, Inc.) was performed on selected isolates. All methods agreed that 26 pairs were indistinguishable and four pairs were different. Discrepant results were as follows: 4 where only SCRA was discordant, 3 where only AB was discordant, 2 where both DL and AB were discordant, and 1 where both DL and SCRA were discordant. All WGM agreed with PFGE. After discrepancy resolution, 80% of the pairs were indistinguishable and 20% were different. A total of 56% of nares results were nonpredictive if negative nares and positive wound cultures are included. Methods agreed 85 to 93% of the time; however, congruence of isolates to a clade was lower. Baseline analysis of types showed that 15 pairs were unique to single patients (30 strains, 38%; 47% of the matching pairs). Twenty-five strains (30%) represented a single clade identical by PFGE, SCRA, and DL, decreasing specificity. Typing method and institutional type frequency are important in assessing MRSA strain relatedness.

A lingual abscess caused by Streptococcus intermedius.

Lingual abscesses are rare. We describe a case in a healthy female with no recent history of trauma. The organism recovered by culture of drainage material collected prior to antibiotic treatment was Streptococcus intermedius, an organism recognized as flora of the oropharynx and associated with abscess formation. The isolate was resistant to clindamycin, which was the antibiotic therapy that the patient received.

Significantly larger numbers of methicillin-resistant Staphylococcus aureus bacteria are recovered from polymicrobial respiratory and wound sites by use of chromogenic primary media than by use of conventional culture.

Previous studies have validated the properties and documented the utility of chromogenic agar for surveillance of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we used one of the chromagars, MRSASelect (Bio-Rad), as one of the primary isolation media for selected wound and respiratory clinical specimens which, in our institution, were typically polymicrobial. We examined a total of 638 specimens; 142 (22%) MRSA isolates were recovered. Twenty-six of these isolates were recovered only on the MRSASelect plate, representing a 28% (15/54) increase for endotracheal aspirates/sputa and a 15% increase for superficial wounds/ulcers (11/73) compared to the results with conventional culture. One isolate (1 CFU) was recovered by conventional medium alone. MRSASelect has generally been used for surveillance cultures; however, we document that an additional 21% of MRSA isolates would have gone unreported in these selected clinical specimens using only standard culture media. For 40% (6/15) of inpatients, MRSA isolated from the MRSASelect plate was the sole indicator of MRSA. Although these isolates can represent either colonization or infection, they are a potential reservoir of infection and nosocomial transmission. Our data support the focused use of chromogenic selective media for the increased detection of MRSA in polymicrobial wound and respiratory specimens, which could have an impact on both clinical treatment and infection control.