Controlled clinical comparison of new pediatric medium with adsorbent polymeric beads (PF Plus) versus charcoal-containing PF medium in the BacT/alert blood culture system.

We conducted a controlled clinical comparison of PF Plus, the new pediatric medium with adsorbent polymeric beads, versus the charcoal-containing PF medium in the BacT/Alert blood culture system. A total of 2,381 pediatric cultures were enrolled, and 1,703 (71.5%) were deemed to be compliant and acceptable for analysis. Seventy-two cultures (4.2%) had a positive result with 80 clinically significant microorganisms. The results showed that the PF Plus medium yielded more clinically significant microorganisms than the BacT/Alert system (P < 0.05). In addition, the PF Plus bottle yielded positive results an average of 5.0 h earlier than the PF bottle for compliant clinically significant cultures (18.3 h versus 23.2 h, P = 0.004). PF Plus is an improved medium for detecting microorganisms that cause pediatric bloodstream infections.

Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices.

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Better tests, better care: improved diagnostics for infectious diseases.

In this IDSA policy paper, we review the current diagnostic landscape, including unmet needs and emerging technologies, and assess the challenges to the development and clinical integration of improved tests. To fulfill the promise of emerging diagnostics, IDSA presents recommendations that address a host of identified barriers. Achieving these goals will require the engagement and coordination of a number of stakeholders, including Congress, funding and regulatory bodies, public health agencies, the diagnostics industry, healthcare systems, professional societies, and individual clinicians.

Bacteremic disseminated tuberculosis in sub-saharan Africa: a prospective cohort study.

BACKGROUND: Disseminated tuberculosis is a major health problem in countries where generalized human immunodeficiency virus (HIV) infection epidemics coincide with high tuberculosis incidence rates; data are limited on patient outcomes beyond the inpatient period. METHODS: We enrolled consecutive eligible febrile inpatients in Moshi, Tanzania, from 10 March 2006 through 28 August 2010; those with Mycobacterium tuberculosis bacteremia were followed up monthly for 12 months. Survival, predictors of bacteremic disseminated tuberculosis, and predictors of death were assessed. Antiretroviral therapy (ART) and tuberculosis treatment were provided. RESULTS: A total of 508 participants were enrolled; 29 (5.7%) had M. tuberculosis isolated by blood culture. The median age of all study participants was 37.4 years (range, 13.6-104.8 years). Cough lasting >1 month (odds ratio [OR], 13.5; P< .001), fever lasting >1 month (OR, 7.8; P = .001), weight loss of >10% (OR, 10.0; P = .001), lymphadenopathy (OR 6.8; P = .002), HIV infection (OR, undefined; P < .001), and lower CD4 cell count and total lymphocyte count were associated with bacteremic disseminated tuberculosis. Fifty percent of participants with M. tuberculosis bacteremia died within 36 days of enrollment. Lower CD4 cell count (OR, 0.88; P = .049) and lower total lymphocyte count (OR, 0.76; P = .050) were associated with death. Magnitude of mycobacteremia tended to be higher among those with lower CD4 cell counts, but did not predict death. CONCLUSIONS: In the era of free ART and access to tuberculosis treatment, almost one half of patients with M. tuberculosis bacteremia may die within a month of hospitalization. Simple clinical assessments can help to identify those with the condition. Advanced immunosuppression predicts death. Efforts should focus on early diagnosis and treatment of HIV infection, tuberculosis, and disseminated disease.

Performance of nucleic acid amplification following extraction of 5 milliliters of whole blood for diagnosis of Mycobacterium tuberculosis bacteremia.

To investigate the performance of a nucleic acid amplification test (NAAT) for the diagnosis of Mycobacterium tuberculosis bacteremia, 5-ml aliquots of blood were inoculated into bioMérieux mycobacterial (MB) bottles and incubated, and 5-ml aliquots of blood were extracted and tested by real-time PCR. Of 25 samples from patients with M. tuberculosis bacteremia, 9 (36.0%) were positive and 1 (1.5%) of 66 control samples was positive by NAAT. The NAAT shows promise, but modifications should focus on improving sensitivity.

Methicillin-susceptible Staphylococcus aureus endocarditis isolates are associated with clonal complex 30 genotype and a distinct repertoire of enterotoxins and adhesins.

BACKGROUND: Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity. METHODS: IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes. RESULTS: 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all). CONCLUSIONS: MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.

Controlled comparison of BacT/Alert MB system, manual Myco/F lytic procedure, and isolator 10 system for diagnosis of Mycobacterium tuberculosis Bacteremia.

We compared the performance of the BacT/Alert MB system, that of the manual Bactec Myco/F Lytic procedure, and that of the Isolator 10 lysis-centrifugation system in the detection of Mycobacterium tuberculosis bacteremia. Mean times to detection were 16.4 days for BacT/Alert MB versus 20.0 days for Myco/F Lytic, 16.5 days for BacT/Alert MB versus 23.8 days for Isolator 10, and 21.1 days for Bactec Myco/F Lytic versus 22.7 days for Isolator 10. There were no significant differences in yields. The mean (range) magnitude of mycobacteremia was 30.0 (0.4, 90.0) CFU/ml and was correlated with the time to positivity in the BacT/Alert MB system (r = -0.4920). M. tuberculosis bacteremia was detected more rapidly in a continuously monitored liquid blood culture system, but the mean time to positivity exceeded 3 weeks.

Quantitation of Candida CFU in initial positive blood cultures.

One potential limitation of DNA-based molecular diagnostic tests for Candida bloodstream infection (BSI) is organism burden, which is not sufficiently characterized. We hypothesized that the number of CFU per milliliter (CFU/ml) present in an episode of Candida BSI is too low for reliable DNA-based diagnostics. In this study, we determined Candida burden in the first positive blood culture and explored factors that affect organism numbers and patient outcomes. We reviewed records of consecutive patients with a positive blood culture for Candida in the lysis-centrifugation blood culture system (Isolator, Wampole Laboratories, Cranbury, NJ) from 1987 to 1991. Descriptive statistics and logistic regression analyses were performed. One hundred fifty-two episodes of Candida BSI were analyzed. Patient characteristics included adult age (72%), indwelling central venous catheters (83%), recent surgery (29%), neutropenia (24%), transplant (14%), and other immune suppression (21%). Rates of treatment success and 30-day mortality for candidemia were each 51%. The median CFU/ml was 1 (mode 0.1, range 0.1 to >1,000). In the multivariate analysis, pediatric patients were more likely than adults to have high organism burdens (odds ratio [OR], 10.7; 95% confidence interval [95% CI], 4.3 to 26.5). Initial organism density did not affect patient outcome. Candida CFU/ml in the first positive blood culture of a BSI episode varies greatly; >50% of cultures had ≤1 CFU/ml, a concentration below the experimental yeast cell threshold for reliable DNA-based diagnostics. DNA-based diagnostics for Candida BSI will be challenged by low organism density and the need for sufficient specimen volume; future research on alternate targets is warranted.

Dissemination of an Enterococcus Inc18-Like vanA plasmid associated with vancomycin-resistant Staphylococcus aureus.

Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.

The (1,3){beta}-D-glucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation.

The Fungitell assay for (1,3)ß-D-glucan (BG) detection in serum has been evaluated in patients with invasive fungal infections (IFIs) and healthy controls and for the early diagnosis of IFI in cancer patients. We evaluated the BG assay for the detection of IFI in lung transplant recipients. Serial serum samples were prospectively collected from patients undergoing lung transplants at Duke Hospital. Fungal infections were classified according to revised European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. A receiver operator characteristic (ROC) curve was generated; possible causes for false-positive and false-negative tests were investigated by linear regression analysis. Seven hundred fifty-six serum specimens from 59 subjects without IFI and 41 specimens from 14 patients with proven or probable IFI were tested. The area under the ROC curve was 0.69. Based on a 60-pg/ml positive cutoff, per-patient sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 64%, 9%, 14%, and 50%, respectively; per-test estimates were 71%, 59%, 9%, and 97%, respectively. The majority (92%) of patients not diagnosed with an IFI had at least one BG level of ≥60 pg/ml, and 90% had at least one BG level of ≥80 pg/ml. Respiratory colonization with mold and hemodialysis significantly affected mean BG levels. In conclusion, the accuracy of the BG test is marginal and its utility as a tool for the early diagnosis of IFI is questionable in the lung transplant population. Although the NPV of the BG test is high, the low PPV limits its utility as a screening tool for early diagnosis of IFI.

Multicenter study to determine disk diffusion and broth microdilution criteria for prediction of high- and low-level mupirocin resistance in Staphylococcus aureus.

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of >or=512 microg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-microg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the "gold standard" for HLR, the sensitivity and specificity of a single-well 256 microg/ml BMD test were 97 and 99%, respectively, and those for the 200-microg disk test were 98 and 99%, respectively. Testing with two disks, 200 microg and 5 microg, was evaluated for its ability to distinguish HLR isolates (MICs >or= 512 microg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 microg/ml), and susceptible isolates (MICs

Correlation of cefoxitin MICs with the presence of mecA in Staphylococcus spp.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of < or = 4 microg/ml for mecA-negative and > or = 6 or 8 microg/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of < or = 2 microg/ml for mecA-negative and > or = 4 microg/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.

Murine typhus and febrile illness, Nepal.

Murine typhus was diagnosed by PCR in 50 (7%) of 756 adults with febrile illness seeking treatment at Patan Hospital in Kathmandu, Nepal. Of patients with murine typhus, 64% were women, 86% were residents of Kathmandu, and 90% were unwell during the winter. No characteristics clearly distinguished typhus patients from those with blood culture-positive enteric fever.

Genotypic diversity of anaerobic isolates from bloodstream infections.

Accurate species determination for anaerobes from blood culture bottles has become increasingly important with the reemergence of anaerobic bacteremia and prevalence of multiple-drug-resistant microorganisms. Our knowledge of the taxonomical diversity of anaerobes that cause bloodstream infections is extremely limited, because identification historically has relied on conventional methods. Over a 5-year period, we profiled anaerobic bacteremia at a large tertiary care hospital with 16S rRNA gene sequencing to gain a better understanding of the taxonomical diversity of the bacteria. Of 316 isolates, 16S rRNA gene sequencing and phylogenetic analysis identified 316 (100%) to the genus or taxonomical group level and 289 (91%) to the species level. Conventional methods identified 279 (88%) to the genus level and 208 (66%) to the species level; 75 (24%) were misidentified at the species level, and 33 (10%) results were inconclusive. High intragenus variability was observed for Bacteroides and Clostridium species, and high intraspecies variability was observed for Bacteroides thetaiotaomicron and Fusobacterium nucleatum. Sequence-based identification has potential benefits in comparison to conventional methods, because it more accurately characterizes anaerobes within taxonomically related clusters and thereby may enable better correlation with specific clinical syndromes and antibiotic resistance patterns.

Multicenter evaluation of the new Vitek 2 Neisseria-Haemophilus identification card.

The new Neisseria-Haemophilus identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily Neisseria species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.

Multicenter evaluation of the Vitek 2 anaerobe and Corynebacterium identification card.

The new anaerobe and Corynebacterium (ANC) identification card for Vitek 2 was compared with a 16S rRNA gene sequencing (16S) reference method for accuracy in the identification of corynebacteria and anaerobic species. Testing was performed on a Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains that were tested 20 times over a minimum of 10 days at all three sites. A challenge set of 50 well-characterized strains and 365 recent fresh and frozen clinical isolates were included in the study. The expected positive and negative biochemical well reactions were also evaluated for substrate reproducibility. All strains were tested with the ANC card, and clinical isolates were saved for 16S rRNA gene sequencing. All reproducibility tests yielded expected results within a 95% confidence interval, except for that with Corynebacterium striatum ATCC 6940, for which identification failed at one trial site. For the challenge isolates, there was 98% correct identification, 5% low discrimination, and 2% incorrect identification, and 0% were unidentified. For clinical strains, there was 95.1% correct identification, 4.9% low discrimination, and 4.6% incorrect identification, and 0.3% were unidentified. The 4.6% (17/365) of clinical isolates that were incorrectly identified consisted of 14 isolates that were correct at the genus level and three that were incorrect at the genus level. The new ANC card met all performance criteria within a 95% confidence interval compared to the identification performance by 16S rRNA gene sequencing.

Association between 16S-23S internal transcribed spacer sequence groups of Mycobacterium avium complex and pulmonary disease.

Organisms within the Mycobacterium avium complex (MAC) may have differential virulence. We compared 33 subjects with MAC pulmonary disease to 75 subjects with a single positive culture without disease. M. avium isolates were significantly more likely to be associated with MAC pulmonary disease (odds ratio = 5.14, 95% confidence interval = 1.25 to 22.73) than M. intracellulare.

Phylogenetic analysis of viridans group streptococci causing endocarditis.

Identification of viridans group streptococci (VGS) to the species level is difficult because VGS exchange genetic material. We performed multilocus DNA target sequencing to assess phylogenetic concordance of VGS for a well-defined clinical syndrome. The hierarchy of sequence data was often discordant, underscoring the importance of establishing biological relevance for finer phylogenetic distinctions.

Genotypic diversity of coagulase-negative staphylococci causing endocarditis: a global perspective.

Coagulase-negative staphylococci (CNS) are important causes of infective endocarditis (IE), but their microbiological profiles are poorly described. We performed DNA target sequencing and susceptibility testing for 91 patients with definite CNS IE who were identified from the International Collaboration on Endocarditis-Microbiology, a large, multicenter, multinational consortium. A hierarchy of gene sequences demonstrated great genetic diversity within CNS from patients with definite endocarditis that represented diverse geographic regions. In particular, rpoB sequence data demonstrated unique genetic signatures with the potential to serve as an important tool for global surveillance.

Venous thrombosis in patients with short- and long-term central venous catheter-associated Staphylococcus aureus bacteremia.

OBJECTIVE: Infection and thrombosis are important complications of intravascular catheters. The purpose of this study was to determine the incidence of thrombosis in patients with central venous catheter-associated Staphylococcus aureus bacteremia and the utility of physical examination for diagnosing upper extremity or neck venous thrombosis. DESIGN: Prospective observational cohort. SETTING: Tertiary care facility. PATIENTS: In all, 65 consecutive patients with catheter-associated S. aureus bacteremia with central venous catheters of the internal jugular, brachial, or subclavian veins were eligible for participation. INTERVENTION: From July 1999 through August 2004, enrolled patients underwent physical examination and ultrasonography independently to identify the presence of catheter-associated thrombosis. Study ultrasonograms were interpreted blindly using defined criteria. Outcomes were defined at 12-wk follow-up. MEASUREMENTS AND MAIN RESULTS: A total of 48 patients were enrolled. By ultrasonography, definite or possible thrombosis was present in 34 of 48 patients (71%) in this cohort. Death or recurrent bacteremia occurred in 11/34 (32%) infected patients with thrombosis and two of 14 (14%) infected patients without thrombosis (p = .29). Sensitivity of all physical examination findings, either alone or in combination, was low (< or = 24%). Only engorged veins upon hand elevation and the presence of multiple physical examination abnormalities were specific (100% each). CONCLUSIONS: Thrombosis is a common complication of central venous catheter-associated S. aureus bacteremia. Patients with central venous catheter-associated S. aureus bacteremia should undergo ultrasonography to detect thromboses even if the physical examination is normal.