Multicenter evaluation of the Sepsityper™ extraction kit and MALDI-TOF MS for direct identification of positive blood culture isolates using the BD BACTEC™ FX and VersaTREK(®) diagnostic blood culture systems.

AIMS: (i) Evaluation of delayed time to blood culture extraction by the Sepsityper kit and impact of shipping pellets off-site for MALDI-TOF MS analysis. (ii) Comparison of Sepsityper and laboratory-developed extraction methods from a literature review. METHODS AND RESULTS: Using two blood culture systems (BD BACTEC and VersaTREK), we extracted 411 positive blood cultures using the Sepsityper kit to mimic a potential protocol for institutions without a MALDI-TOF MS. Extracted pellets were shipped and analysed on the Bruker UltraflexIII. Successful extraction of 358 (87·1%) samples was determined by the presence of detectable proteins. MALDI-TOF MS correctly identified 332 (80·8%) samples. CONCLUSIONS: Delayed time to extraction did not affect Sepsityper extraction or MALDI-TOF MS accuracy. The extracted pellets remain stable and provide accurate results by MALDI-TOF MS when shipped at room temperature to off-site reference laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to show that institutions without a MALDI-TOF MS can take advantage of this innovative technology by shipping a volume of blood to an off-site laboratory for extraction and MALDI-TOF MS analysis. We also performed a literature review to compare various extraction methods.

Prevalence of Trichomonas vaginalis and coinfection with Chlamydia trachomatis and Neisseria gonorrhoeae in the United States as determined by the Aptima Trichomonas vaginalis nucleic acid amplification assay.

Our aim was to determine Trichomonas vaginalis prevalence using the Aptima Trichomonas vaginalis assay (ATV; Gen-Probe) and the prevalence of Chlamydia trachomatis and Neisseria gonorrhoeae coinfections in U.S. women undergoing screening for C. trachomatis/N. gonorrhoeae. Discarded urogenital samples from 7,593 women (18 to 89 years old) undergoing C. trachomatis/N. gonorrhoeae screening using the Aptima Combo 2 assay (Gen-Probe) in various clinical settings were tested with ATV. Overall, T. vaginalis, C. trachomatis, and N. gonorrhoeae prevalences were 8.7%, 6.7%, and 1.7%, respectively. T. vaginalis was more prevalent than C. trachomatis or N. gonorrhoeae in all age groups except the 18- to 19-year-old group. The highest T. vaginalis prevalence was in women ≥ 40 years old (>11%), while the highest C. trachomatis prevalence (9.2%) and N. gonorrhoeae prevalence (2.2%) were in women <30 years old. Coinfection prevalences were 1.3% for C. trachomatis/T. vaginalis, 0.61% for C. trachomatis/N. gonorrhoeae and N. gonorrhoeae/T. vaginalis, and 0.24% for C. trachomatis/N. gonorrhoeae/T. vaginalis and highest in women <30 years old. T. vaginalis prevalence differed by race/ethnicity, with the highest prevalence in black women (20.2%). T. vaginalis prevalence ranged from 5.4% in family planning clinics to 22.3% in jails. Multivariate analysis determined that ages of ≥ 40 years, black race, and patient locations were significantly associated with T. vaginalis infection. T. vaginalis is the most common sexually transmitted infection (STI) in women of >40 years, while C. trachomatis and N. gonorrhoeae prevalence is lowest in that age group. Higher T. vaginalis prevalence in women of >40 years is probably attributed to the reason for testing, i.e., symptomatic status versus routine screening in younger women. Coinfections were relatively low. High T. vaginalis prevalence in all age groups suggests that women screened for C. trachomatis/N. gonorrhoeae, whether asymptomatic or symptomatic, should be screened for T. vaginalis.

Evaluation of a new selective medium, BD BBL CHROMagar MRSA II, for detection of methicillin-resistant Staphylococcus aureus in different specimens.

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.

Diagnostic test accuracy of the BioFire® FilmArray® meningitis/encephalitis panel: a systematic review and meta-analysis.

BACKGROUND: The FilmArray® meningitis/encephalitis (ME) panel is a multiplex PCR assay which can detect the most commonly identified pathogens in central nervous system infections. It significantly decreases the time to diagnosis of ME and data has yielded several positive outcomes. However, in part, reports of both false positive and false negative detections have resulted in concerns about adoption. OBJECTIVES: The aim was to evaluate the ME panel in a diagnostic test accuracy review. DATA SOURCES: The PubMed and EMBASE databases were systematically searched through May 2019. STUDY ELIGIBILITY CRITERIA: Eligible studies were those providing sensitivity and specificity data for the ME panel compared with a reference standard. Studies providing details on false positive and false negative results of the panel as well as further investigation (adjudication) of the discordant results between the panel and comparator assays were included and assessed separately. PARTICIPANTS: Patients with suspected ME for whom a panel was ordered were included. METHODS: The ME panel was compared to reference standard methods for diagnosing community-acquired ME. We performed a meta-analysis and calculated the summary sensitivity and specificity of the ME panel. Moreover, we evaluated the false positive and false negative results of the panel. RESULTS: Thirteen studies (3764 patients) were included in the review and 8 of them (3059 patients) were pooled in a meta-analysis. The summary estimates of sensitivity and specificity with 95% confidence intervals (CI) was 90% (95% CI 86-93%) and 97% (95% CI 94-99%), respectively. When we looked specifically at studies that assessed further the false positive and false negative results, false positive detections were 11.4% and 4% before and after adjudication, respectively. The highest proportion of false positive was observed for Streptococcus pneumoniae followed by Streptococcus agalactiae. False negative isolates were 2.2% and 1.5% before and after adjudication, respectively. Herpes simplex virus 1 and 2, enterovirus and Cryptococcus neoformans/gattii had the highest proportions of false negative determinations. False negative C. neoformans/gattii were mostly patients with positive antigen titres, on treatment or cleared disease. CONCLUSIONS: The currently available literature suggests that the ME panel has high diagnostic accuracy. However, the decision for implementation should be individualized based on the needs of the patient population, the capabilities of the laboratory, and the knowledge of the healthcare providers that will utilize the test.

Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays.

A multicenter preclinical evaluation was conducted to evaluate the performance of two Cepheid Xpert assays for detection of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus. Sensitivity was 97.1% and 98.3% for MRSA in wound and blood culture specimens, respectively. Sensitivity was 100% for S. aureus from both specimen types.

Receptors for the carboxyl-terminal region of pth(1-84) are highly expressed in osteocytic cells.

PTH is a potent systemic regulator of cellular differentiation and function in bone. It acts upon cells of the osteoblastic lineage via the G protein-coupled type-1 PTH/PTH-related peptide receptor (PTH1R). Carboxyl fragments of intact PTH(1-84) (C-PTH fragments) are cosecreted with it by the parathyroid glands in a calcium-dependent manner and also are generated via proteolysis of the hormone in peripheral tissues. Receptors that recognize C-PTH fragments (CPTHRs) have been described previously in osteoblastic and chondrocytic cells. To directly study CPTHRs in bone cells, we isolated clonal, conditionally transformed cell lines from fetal calvarial bone of mice that are homozygous for targeted ablation of the PTH1R gene and transgenically express a temperature-sensitive mutant SV40 T antigen. Cells with the highest specific binding of the CPTHR radioligand (125)I-[Tyr(34)]hPTH(19-84) exhibited a stellate, dendritic appearance suggestive of an osteocytic phenotype and expressed 6- to 10-fold more CPTHR sites/cell than did osteoblastic cells previously isolated from the same bones. In these osteocytic (OC) cells, expression of mRNAs for CD44, connexin 43, and osteocalcin was high, whereas that for alkaline phosphatase and cbfa-1/osf-2 was negligible. The CPTHR radioligand was displaced completely by hPTH(1-84), hPTH(19-84) and hPTH(24-84) (IC(50)s = 20-50 nM) and by hPTH(39-84) (IC(50) = 500 nM) but only minimally (24%) by 10,000 nM hPTH(1-34). CPTHR binding was down-regulated dose dependently by hPTH(1-84), an effect mimicked by ionomycin and active phorbol ester. Human PTH(1-84) and hPTH(39-84) altered connexin 43 expression and increased apoptosis in OC cells. Apoptosis induced by PTH(1-84) was blocked by the caspase inhibitor DEVD. We conclude that osteocytes, the most abundant cells in bone, may be principal target cells for unique actions of intact PTH(1-84) and circulating PTH C-fragments that are mediated by CPTHRs.

Rescue of the skeletal phenotype of vitamin D receptor-ablated mice in the setting of normal mineral ion homeostasis: formal histomorphometric and biomechanical analyses.

1,25-Dihydroxyvitamin D3 has been shown to play an important role in vitro in regulating osteoblast gene transcription and promoting osteoclast differentiation. To address the role of the vitamin D receptor (VDR) in skeletal homeostasis, formal histomorphometric analyses were performed in VDR null mice in the setting of impaired mineral ion homeostasis as well as in VDR null mice in whom normal mineral ion homeostasis had been preserved. In hypocalcemic VDR null mice, there was an increase in bone volume as a result of a dramatic increase in osteoid. There was also an increase in the number of osteoblasts without a significant change in the number of osteoclasts. Examination of the growth plate revealed marked disorganization, with an increase in vascularity and matrix. Biomechanical parameters demonstrated increased bone fragility in the hypocalcemic VDR null mice. In the VDR ablated mice in whom normal mineral ion homeostasis had been preserved, none of these measurements was significantly different from those in wild-type littermates raised under identical conditions. Notably, the morphology and width of the growth plate were indistinguishable from those in wild-type controls, demonstrating that a calcium/phosphorus/lactose-enriched diet started at 16 days of age in the VDR null mice permits the development of both normal morphology in the growth cartilage and adjacent metaphysis and normal biomechanical competence of cortical bone. Thus, the principle action of the VDR in skeletal growth, maturation, and remodeling is its role in intestinal calcium absorption. The skeletal consequences of VDR ablation are a result of impaired intestinal calcium absorption and/or the resultant secondary hyperparathyroidism and hypophosphatemia.

Deficient mineralization of intramembranous bone in vitamin D-24-hydroxylase-ablated mice is due to elevated 1,25-dihydroxyvitamin D and not to the absence of 24,25-dihydroxyvitamin D.

The 25-hydroxyvitamin D-24-hydroxylase enzyme (24-OHase) is responsible for the catabolic breakdown of 1,25-dihydroxyvitamin D [1,25(OH)2D], the active form of vitamin D. The 24-OHase enzyme can also act on the 25-hydroxyvitamin D substrate to generate 24,25-dihydroxyvitamin D, a metabolite whose physiological importance remains unclear. We report that mice with a targeted inactivating mutation of the 24-OHase gene had impaired 1,25(OH)2D catabolism. Surprisingly, complete absence of 24-OHase activity during development leads to impaired intramembranous bone mineralization. This phenotype was rescued by crossing the 24-OHase mutant mice to mice harboring a targeted mutation in the vitamin D receptor gene, confirming that the elevated 1,25(OH)2D levels, acting through the vitamin D receptor, were responsible for the observed accumulation of osteoid. Our results confirm the physiological importance of the 24-OHase enzyme for maintaining vitamin D homeostasis, and they reveal that 24,25-dihydroxyvitamin D is a dispensable metabolite during bone development.

Difference in reaction time between subjects with schizotypal and borderline personality disorders.

Patients with schizophrenic spectrum disorders and affective disorders were compared on a reaction time procedure. The nonhospitalized schizotypal subjects performed similarly to the schizophrenic patients on the crossover measure. Mean reaction time discriminated between hospitalized and nonhospitalized patients rather than between types of pathology.

Investigation of the associative network in schizophrenia using the semantic priming paradigm.

The semantic priming version of a lexical decision task was utilized in order to distinguish between automatic and controlled attentional processes in schizophrenia. 12 schizophrenic patients, 12 psychiatric control patients, and 12 normal subjects completed this lexical decision task. Although the overall reaction times were longer for the schizophrenic subjects, all groups manifested semantic facilitation on the lexical decision task. The presence of semantic facilitation in schizophrenic patients supports the theory that automatic attentional processing is intact in schizophrenia.

Activation and facilitation in the lexicon of schizophrenics.

45 schizophrenic patients of three different subtypes (chronic undifferentiated, schizoaffective and paranoid) and 15 normal control subjects completed a semantic priming version of the lexical decision task. Schizophrenic subjects demonstrated longer reaction times relative to normal controls; however, all of the subjects showed semantic facilitation effects. No significant differences in reaction times were found among the three schizophrenic subtype groups. These results suggest that initial sensory-perceptual processing is unimpaired in schizophrenia and thus provide evidence of intact automatic processing. The similarity in performance among the schizophrenic subtype groups refutes the theory of a paranoid/non-paranoid dimension at the initial level of automatic processing.

Susceptibility of Haemophilus influenzae to amoxicillin/clavulanic acid, erythromycin, cefaclor, and trimethoprim/sulfamethoxazole.

A total of 126 strains of Haemophilus influenzae were examined for susceptibility to amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefaclor, and erythromycin by an agar dilution procedure. Fifty strains (eight type B, 42 non-type B), all with ampicillin minimal inhibitory concentrations (MIC) of greater than or equal to 6.2 micrograms/ml, produced beta-lactamase. The remaining 76 strains (18 type B, 59 non-type B) were beta-lactamase-negative. All of these strains had ampicillin MICs of less than or equal to 0.8 micrograms/ml. The combination of amoxicillin and clavulanic acid (2:1) was highly active against all strains tested. With the exception of two strains with amoxicillin/clavulanic acid MICs of 1.6/0.8 ug/ml, all strains were inhibited by concentrations of less than or equal to 0.8/0.4 ug/ml. Trimethoprim/sulfamethoxazole was also found to be highly active (MICs uniformly less than or equal to 0.1/1.9 ug/ml). Cefaclor and erythromycin were the least active of the agents tested. Fourteen strains (10.6%) had cefaclor MICs of greater than 32 ug/ml. Forty-seven strains (35.6%) had erythromycin MICs of greater than 8 micrograms/ml. With the exception of amoxicillin/clavulanic acid beta-lactamase production did not seem to influence the activity of any of the antimicrobials tested. Minimum inhibitory concentrations of amoxicillin/clavulanic acid, although still well within achievable serum levels, were approximately one twofold dilution higher with beta-lactamase-producing H. influenzae type B strains than with beta-lactamase-negative strains.

Impact of CLIA 88 on the clinical microbiology laboratory.

The Clinical Laboratory Improvement Amendments (CLIA 88) to the Clinical Laboratory Improvement Act of 1967 continues to undergo transformation since its implementation more than 2 years ago. The law and its subsequent regulatory modifications were intended to promote high quality in and accurate results from laboratory testing procedures, regardless of the site at which testing occurred. A number of federal regulatory agencies and committees such as the Healthcare Financing Administration, the Clinical Improved Amendments Committee, and the Commission on Laboratory Accreditation, as well as numerous new or modified regulations and requirements have gained importance since CLIA 88 was enacted. In this discussion, components of CLIA 88 that have the greatest impact on clinical microbiology laboratories are presented. In addition, the potential future significance of CLIA 88 are outlined.

Determination of biotypes of Haemophilus influenzae and Haemophilus parainfluenzae a comparison of methods and a description of a new biotype (VIII) of H. parainfluenzae.

A total of 180 strains of Haemophilus influenzae and 119 strains of Haemophilus parainfluenzae were characterized with respect to biotype (i.e., production of indole, urease, and ornithine decarboxylase) using conventional biochemical methods and two commercially available biotyping systems: Trio-Tube Haemophilus system (Carr Microbiologicals) and the Rapid NH System (Inovative Diagnostic Systems). Concordance between the results of the Trio-Tube system and conventional biochemicals was achieved with 294 of the 299 test organisms (98.3%). With the Rapid NH System, concordance with the results of conventional biochemical tests was observed with 275 of the 299 tests strains (92.0%). One previously unrecognized biotype of H. parainfluenzae, designated biotype VIII, is described. Typical reactions of this biotype include indole production but no production of urease or ornithine decarboxylase.

A new classification and diagnostic criteria for invasive fungal sinusitis.

OBJECTIVE: To develop criteria for the diagnosis of invasive fungal sinusitis. DESIGN: Review of the literature on invasive fungal sinusitis in the context of a population of 30 patients with fungal sinusitis and 24 patients with chronic bacterial sinusitis. SETTING: Tertiary care medical center. RESULTS: Our review revealed no consensus in the literature on the classification of the syndromes of invasive fungal sinusitis and no criteria for their diagnosis. Moreover, the existing syndromes of invasive fungal sinusitis lacked specificity and one of the more commonly cited syndromes, primary aspergillosis of the paranasal sinuses, is a granulomatous disease that occurs rarely outside Africa. Two of our 30 patients with fungal sinusitis had a previously unrecognized form of invasive disease. Both were middle-aged adults with well-controlled type 2 diabetes mellitus, apical orbital syndrome, and a similar course: proptosis resulting from fungal expansion out of an ethmoid sinus, a protracted illness of 6 months or longer, visual changes, late neurological symptoms reflecting cavernous sinus invasion, and death. The syndrome in these 2 patients is distinct from the syndrome of fulminant invasive fungal sinusitis, (eg, mucormycosis) with nasal eschar, intracerebral fungal dissemination by vascular invasion, and death in days, and the granulomatous form. CONCLUSIONS: We conclude that there are 3 forms of invasive fungal sinusitis and propose that they be termed (1) granulomatous, (2) acute fulminant, and (3) chronic invasive. The latter category reflects the syndrome seen in our 2 patients. Furthermore, the following 2 diagnostic criteria for invasive fungal sinusitis are proposed: (1) sinusitis confirmed by radiological imaging and (2) histopathological evidence of hyphal forms within sinus mucosa, submucosa, blood vessels, or bone. The specificity of hyphae within sinus mucosa for tissue invasion was supported by the absence of stainable hyphae in the mucosa of patients with chronic bacterial sinusitis or in the mucosa of our described patients with allergic fungal sinusitis and mycetoma.

Is Music Therapy an Effective Intervention for Dementia?A Meta-Analytic Review of Literature.

A recent qualitative review of literature in the area of music/ music therapy and dementias published since 1985 suggested that music/music therapy is an effective intervention for maintaining and improving active involvement, social, emotional and cognitive skills, and for decreasing behavioral problems of individuals with dementias (Brotons, Koger, & Pickett-Cooper, 1997). The present analysis sought to update and quantify this relationship, and investigate the extent to which methodological variables influenced treatment effectiveness. Twenty-one empirical studies, with a total of 336 subjects suffering from symptoms of dementia, were included in the meta-analysis. Overall, the effect of music/music therapy was found to be highly significant. A homogeneity analysis determined that the effect sizes were not consistent across studies; thus, a series of moderating variable analyses were conducted. We were unable to determine the source of variability between studies by analyzing type of therapeutic intervention (active or passive), music (live or taped), therapist's training (trained music therapist vs. other professional), dependent variable (behavioral, cognitive, or social), or length of treatment. Although the published literature demonstrates that music/music therapy is an effective method overall for treating symptoms of dementia, systematic variation of treatment protocols is necessary to identify the underlying mechanisms and delineate the most effective techniques.

Oseltamivir-resistant 2009-2010 pandemic influenza A (H1N1) in an immunocompromised patient.

Although neuraminidase inhibitors are active against most 2009-2010 pandemic influenza A (H1N1) swine-origin strains, sporadic cases of oseltamivir resistance have been described. Since April 2009, 54 cases of oseltamivir-resistant H1N1 swine-origin have been reported in the USA (http://www.cdc.gov/flu/weekly/; accessed 1 February 2010). Approximately 1.4% of tested isolates are oseltamivir resistant. We report a patient with an underlying hematological malignancy who was hospitalized with influenza A (H1N1) swine-origin and whose strain developed oseltamivir resistance during therapy.