Implementing Alfred60AST in a clinical lab: Clinical impact on the management of septic patients and financial analysis.

INTRODUCTION: Sepsis is an important cause of morbidity and mortality. An accelerated microbiology diagnosis is crucial in order to reduce the time to initiate targeted antibiotic therapy. The Alfred60AST system is able to provide phenotypic Antimicrobial Susceptibility Testing (AST) results within hours. This study has two objectives: assess the clinical impact of this technology and determine its cost-effectiveness. METHODS: During a ten-week period, all new enterobacterial or enterococcal bloodstream infection was analyzed with the Alfred60AST system, in parallel with routine methods. Its impact on the clinician's therapeutic strategy was studied. In order to assess the financial and practical aspects of the method, an analysis of the extracosts and a survey of the technical staff were conducted. RESULTS: Fifty-three cases of bacteriemia were included. For the Enterobacteriaceae bacteriemias, a clinical impact was shown in 18.9% of the cases (e.g, treatment modification). The financial analysis highlighted an increase in costs (+38% for Enterobacteriaceae, +50% for Enterococci), compared to the theoretical costs reported by the firm, due to the workflow and the volumes of samples used. Finally, results of the technical staff survey were favorable in terms of ease of use of the system. CONCLUSION: In addition to its ease of use, the Alfred60AST system is able to provide an AST in a record time. This study shows a real interest of the technique in the therapeutic management of patients with enterobacterial sepsis. However, its routine implementation requires an increase of the analyzed volumes as well as a 24/7 organization of the laboratory in order to be profitable.

Optimal Detection of Latent Mycobacterium tuberculosis Infection by Combined Heparin-Binding Hemagglutinin (HBHA) and Early Secreted Antigenic Target 6 (ESAT-6) Whole-Blood Interferon Gamma Release Assays.

Optimal detection of latent tuberculosis (TB) infection (LTBI) remains a challenge, although it is essential to reach the goal of TB elimination. Our objective was to develop and clinically evaluate a user-friendly, 24-h, whole-blood (WB) interferon gamma (IFN-γ) release assay (IGRA) improving the detection of LTBI, compared to available tests. One milliliter of blood was divided into four aliquots and in vitro stimulated for 24 h with two different stage-specific mycobacterial antigens, i.e., heparin-binding hemagglutinin (HBHA) and early secreted antigenic target 6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively, in addition to positive and negative controls. Clinical evaluation was performed on two independent cohorts of carefully selected subjects, i.e., a training cohort of 83 individuals and a validation cohort of 69 individuals. Both cohorts comprised LTBI subjects (asymptomatic people with a positive tuberculin skin test result and potential exposure to TB index cases), patients with active TB (aTB), and noninfected controls. The sensitivity and specificity of the WB-HBHA-IGRA to identify LTBI subjects among asymptomatic individuals were 93%. Combining the results in response to HBHA and ESAT-6 allowed us to identify LTBI subgroups. One group, with IFN-γ responses to HBHA only, was easily differentiated from patients with aTB. The other group, responding to both antigens like the aTB group, is likely at risk to reactivate the infection and should be prioritized for prophylactic anti-TB treatment. The combined WB-IGRA may be offered to clinicians for the selection of LTBI subjects to benefit from prophylactic treatment.

Antimicrobial susceptibility testing determined by Alfred 60/AST (Alifax®) in a multi-sites lab: performance's evaluation and optimization of workflow.

PURPOSE: New techniques are needed to speed-up the identification and antimicrobial susceptibility testing (AST) of bacteria associated with bloodstream infections. Alfred 60/AST (Alifax®, Polverara, Italy) performs AST by light scattering directly from positive blood cultures. METHODS: We evaluated Alfred 60/AST performances for 4 months. Each new episode of bacteraemia was included and AST were compared to either our rapid automated AST (Vitek® 2) or disk diffusion method. The discrepancies were investigated using Etest®. The time-to-result (TTR) was evaluated by comparing the blood volume inserted into Alfred 60/AST, i.e. 2 versus 7 blood drops. Taking into account the TTR, the workflow of positive blood cultures and the availability of AST results was studied in order to optimize the implementation of Alfred 60/AST. RESULTS: A total of 249 samples and 1108 antibiotics for AST were tested. After exclusion of unavailable results, 1008 antibiotics were analysed. 94.9% (n = 957/1008) of the antibiotics showed categorical agreement. There were 14 very major errors (VME), 24 major errors (ME) and 13 minor errors (mE). The VME were mostly related to clindamycin (64.3%) whereas meropenem and piperacillin-tazobactam constituted the major part (37.5% and 61.5%) of ME and mE respectively. Results were highly reliable for Enterobacterales and enterococci. The mean TTR ranged between 4.3 and 6.3 h and was statistically 20 min faster when applying the 7 blood drops protocol. We showed that Alfred 60/AST could give relievable results within working hours for positive blood culture which are flagged the same day between 12:00 am and 12:00 pm. CONCLUSION: Our study confirmed that Alfred 60/AST gives reliable AST results in a short period of time, especially for Enterobacterales and enterococci. AST could thus be easily obtained the same day of a positive blood culture. Clinical impact studies are mandatory to validate a 24/24 working.

Predictive Factors of In-Hospital Mortality in Older Adults with Community-Acquired Bloodstream Infection.

OBJECTIVES: To assess the prevalence of intra-hospital mortality and associated risk factors in older people aged 75+, admitted with blood stream infections (BSI). DESIGN: Single center retrospective study performed in an 850-bed of the academic hospital of the Université Libre de Bruxelles. SETTING AND PARTICIPANTS: From January 2015 to December 2017, all inpatients over 75 years old admitted with BSI were included. MEASURES: Demographical, clinical and microbiological data were collected. RESULTS: 212 patients were included: median age was 82 [79-85] years and 60 % were female. The in-hospital mortality rate was 19%. The majority of microorganisms were Gram-negative strains, of which Escherichia coli was the most common, and urinary tract infection was the most common origin of BSI. Compared to patients who survived, the non-survivor group had a higher SOFA score (6 versus 3, p<0.0001), a higher comorbidity score (5 versus 4, p<0.0001), more respiratory tract infections (28 vs 6 %, p < 0.0001) and fungal infections (5 vs 1 %, p = 0.033), bedridden status (60 vs 25 %, p < 0.0001), and healthcare related infections (60 vs 40 %, p = 0.019). Using Cox multivariable regression analysis, only SOFA score was independently associated with mortality (HR 1.75 [95%IC 1.52-2.03], p<0.0001). CONCLUSIONS AND IMPLICATIONS: BSI in older people are severe infections associated with a significant in-hospital mortality. Severity of clinical presentation at onset remains the most important predictor of mortality for BSI in older people. BSI originating from respiratory source and bedridden patients are at greater risk of intra-hospital mortality. Further prospective studies are needed to confirm these results.

Investigation of unbound colistin A and B in clinical samples using a mass spectrometry method.

Colistin, used as a last-resort drug, has a narrow therapeutic range that justifies therapeutic drug monitoring. Few data are available in the literature regarding the in vivo unbound fraction of colistin. The objectives of this study were to develop a method to isolate unbound colistin in clinical samples by ultrafiltration and to quantify it. The association between unbound colistin and biological parameters (total protein, albumin, alpha-1-acid glycoprotein and creatinine) was investigated. The measured ranges were 0.036-7.160 mg/L for colistin A and 0.064-9.630 mg/L for colistin B. The process of isolation and determination of unbound colistin was applied to clinical samples (n = 30) within 40 min and no non-specific binding was observed during the ultracentrifugation step. The median unbound fractions of colistin measured were 34.3% (12.8-51.0%) and 53.4% (27.0-77.8%) for colistin A and B, respectively. High interindividual biological variation of binding was observed for colistin A and B that was not explained by the biochemical parameters studied. The method developed could be useful to improve outcomes for patients.

Prospective evaluation of a high multiplexing real-time polymerase chain reaction array for the rapid identification and characterization of bacteria causative of nosocomial pneumonia from clinical specimens: a proof-of-concept study.

The purpose of this study was evaluation of the VAPChip assay based on the "Rapid-Array-PCR-technology" which targets 13 respiratory pathogens and 24 ß-lactam resistance genes directly on respiratory clinical specimens. The first step included analysis of 45 respiratory specimens in order to calibrate and determine the threshold for target genes. The second prospective step involved 85 respiratory samples from patients suspected of nosocomial pneumonia collected in two academic hospitals over an 8-month period. Results of the VAPChip assay were compared to routine methods. The first step showed a large proportion of positive signals for H. influenzae and/or S. pneumoniae. For identification, discrepancies were observed in seven samples. Thresholds were adapted and two probes were re-designed to create a new version of the cartridge. In the second phase, sensitivity and specificity of the VAPchip for bacterial identification were 72.9% and 99.1%, respectively. Seventy (82%) pathogens were correctly identified by both methods. Nine pathogens detected by the VAPChip were culture negative and 26 pathogens identified by culture were VAPChip negative. For resistance mechanisms, 11 probes were positive without identification of pathogens with an antimicrobial-susceptibility testing compatible by culture. However, the patient's recent microbiological history was able to explain most of these positive signals. The VAPChip assay simultaneously detects different pathogens and resistance mechanisms directly from clinical samples. This system seems very promising but the extraction process needs to be automated for routine implementation. This kind of rapid point-of-care automated platform permitting a syndromic approach will be the future challenge in the management of infectious diseases.

Culture-Based Methods and Molecular Tools for Azole-Resistant Aspergillus fumigatus Detection in a Belgian University Hospital.

Azole-resistant Aspergillus fumigatus is an increasing worldwide problem with major clinical implications. Surveillance is warranted to guide clinicians to provide optimal treatment to patients. To investigate azole resistance in clinical Aspergillus isolates in our institution, a Belgian university hospital, we conducted a laboratory-based surveillance between June 2015 and October 2016. Two different approaches were used: a prospective culture-based surveillance using VIPcheck on unselected A. fumigatus (n = 109 patients, including 19 patients with proven or probable invasive aspergillosis [IA]), followed by molecular detection of mutations conferring azole resistance, and a retrospective detection of azole-resistant A. fumigatus in bronchoalveolar lavage fluid using the commercially available AsperGenius PCR (n = 100 patients, including 29 patients with proven or probable IA). By VIPcheck, 25 azole-resistant A. fumigatus specimens were isolated from 14 patients (12.8%). Of these 14 patients, only 2 had proven or probable IA (10.5%). Mutations at the cyp51A gene were observed in 23 of the 25 A. fumigatus isolates; TR34/L98H was the most prevalent mutation (46.7%), followed by TR46/Y121F/T289A (26.7%). Twenty-seven (27%) patients were positive for the presence of Aspergillus species by AsperGenius PCR. A. fumigatus was detected by AsperGenius in 20 patients, and 3 of these patients carried cyp51A mutations. Two patients had proven or probable IA and cyp51A mutation (11.7%). Our study has shown that the detection of azole-resistant A. fumigatus in clinical isolates was a frequent finding in our institution. Hence, a rapid method for resistance detection may be useful to improve patient management. Centers that care for immunocompromised patients should perform routine surveillance to determine their local epidemiology.

When you can't see the wood for the trees. Mucor circinelloides: A rare case of primary cutaneous zygomycosis.

A patient with refractory diffuse lymphoma treated for pulmonary invasive aspergillosis developed a concomitant primary cutaneous mucormycosis. The mucormycete was identified by sequencing as Mucor circinelloides. This case confirms the importance of a rapid pathogen diagnosis in immunocompromised patients and the usefulness of molecular methods for identification of rare fungal species.

Elevated ß-lactam concentrations associated with neurological deterioration in ICU septic patients.

BACKGROUND: Although ß-lactams are considered to have a safe therapeutic profile, neurotoxicity has been reported. The aim of this study was to assess the association between ß-lactam concentrations and neurological alterations in septic ICU patients. METHODS: Retrospective study on all ICU patients who were treated with meropenem (MEM), piperacillin-tazobactam (TZP) or ceftazidime/cefepime (CEF) and in whom at least one ß-lactam trough concentration (C min) was determined. Drug levels were measured using high-performance liquid chromatography; C min was normalized to the clinical breakpoint of Pseudomonas aeruginosa (as determined by EUCAST) for each drug (C min/MIC). Changes in neurological status were evaluated using changes in the neurological sequential organ failure assessment score (ΔnSOFA) using the formula: ΔnSOFA = nSOFA(day of TDM) - nSOFA(ICU admission). Worsening neurological status (NWS) was defined as a ΔnSOFA ≥ 1 for an nSOFA on admission of 0-2. RESULTS: We collected 262 C min in 199 patients (130 MEM, 85 TZP, 47 CEF). Median APACHE II score and GCS on admission were 17 and 15, respectively. Overall ICU mortality was 27 %. There were no differences in the occurrence of NWS between antibiotics (39% for MEM, 32% for TZP and 35% for CEF). The occurrence of NWS increased with increasing C min/MIC ranges (P = 0.008); this correlation was found for TZP (P = 0.05) and MEM (P = 0.01), but not for CEF. C min/MIC was an independent predictive factor for NWS (OR 1.12 [1.04-1.20]). CONCLUSION: We found a correlation between high ß-lactam trough concentrations and increased occurrence of neurological deterioration in septic ICU patients. Although our data cannot determine causality, monitoring of ß-lactam levels should be considered when deterioration of neurological status occurs during critical illness.

Broad-spectrum ß-lactams in obese non-critically ill patients.

OBJECTIVES: Obesity may alter the pharmacokinetics of ß-lactams. The goal of this study was to evaluate if and why serum concentrations are inadequate when standard ß-lactam regimens are administered to obese, non-critically ill patients. SUBJECTS AND METHODS: During first year, we consecutively included infected, obese patients (body mass index (BMI) ⩾30 kg m(-2)) who received meropenem (MEM), piperacillin-tazobactam (TZP) or cefepime/ceftazidime (CEF). Patients with severe sepsis or septic shock, or those hospitalized in the intensive care unit were excluded. Serum drug concentrations were measured twice during the elimination phase by high-performance liquid chromatography. We evaluated whether free or total drug concentrations were >1 time (fT>minimal inhibition concentration (MIC)) or >4 times (T>4MIC) the clinical breakpoints for Pseudomonas aeruginosa during optimal periods of time: ⩾40% for MEM, ⩾50% for TZP and ⩾70% for CEF. RESULTS: We included 56 patients (median BMI: 36 kg m(-2)): 14 received MEM, 31 TZP and 11 CEF. The percentage of patients who attained target fT>MIC and T>4MIC were 93% and 21% for MEM, 68% and 19% for TZP, and 73% and 18% for CEF, respectively. High creatinine clearance (107 (range: 6-398) ml min(-1)) was the only risk factor in univariate and multivariate analyses to predict insufficient serum concentrations. CONCLUSIONS: In obese, non-critically ill patients, standard drug regimens of TZP and CEF resulted in insufficient drug concentrations to treat infections due to less susceptible bacteria. Augmented renal clearance was responsible for these low serum concentrations. New dosage regimens need to be explored in this patient population (EUDRA-CT: 2011-004239-29).

Therapeutic drug monitoring of posaconazole in patients with acute myeloid leukemia or myelodysplastic syndrome.

Posaconazole is a broad-spectrum triazole antifungal available as an oral suspension. Pharmacokinetic data showed a high variability of plasma posaconazole concentrations (PPCs) in patients, suggesting a potential interest in drug monitoring. The aim of our prospective study was to measure the PPCs in prophylactically treated patients to evaluate the impact of different factors on these concentrations. In 40 patients treated prophylactically with posaconazole for acute myeloid leukemia or myelodysplastic syndrome between February 2009 and August 2010, PPCs were measured at day 7 of treatment and then twice weekly. Demographic data, clinical data (including gastrointestinal disorders, comedications, and treatment compliance), caloric and fat intake, and biological data were collected and evaluated. We obtained 275 measurements of PPCs, with a median of 430 ng/ml. PPCs were significantly lower in patients with mucositis (P < 0.001), nausea (P = 0.03), diarrhea (P = 0.03), or vomiting (P = 0.05). PPCs were higher in patients with a higher caloric intake (P = 0.02), while the proportion of fat intake had no influence on PPCs (P = 0.84). The concomitant use of proton pump inhibitors decreased the PPCs (P = 0.02), while the use of tacrolimus increased the PPC (P = 0.03). In the multivariate analysis, the factors influencing the PPCs independently were the concomitant use of tacrolimus (P < 0.001), the presence of mucositis (P = 0.01), and food intake (P = 0.02). Our study confirmed the high variability of posaconazole bioavailability and showed the significant influence of gastrointestinal disorders, food intake, and concomitant medication on the PPCs. However, the optimal PPCs still remain to be defined and correlated with clinical efficacy.

An illustrated case of altered cellular immunity after autologous stem cell transplantation.

We report an unusual association of T-cell lymphoma, autologous stem cell transplantation and Progressive Multifocal Leukoencephalopathy.

Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum.

A micro-ELISA, using horseradish peroxidase-conjugated anti-canine IgE and polystyrene microtitration wells for detection of allergen-specific IgE in canine serum, was developed. Specificity of anti-canine IgE was confirmed by reversed cutaneous anaphylaxis evaluations, gel-precipitation reactions, immunoelectrophoresis, immunoaffinity chromatography, and heat inactivation. Individual allergen blanks were used to account for variable nonspecific binding among various allergens, and results were normalized using 4 reference sera. Coefficients of variation for intra-assay and interassay variability ranged from 0.77 to 5.66% and 3.15 to 9.83%, respectively. Results observed with wells coated with mixtures of various allergen extracts yielded results approximately equal to results (average) of wells containing individual components. Agreement between ELISA and skin test results ranged from 43 to 64%, depending on allergen used.

Effect of immunotherapy on the serum concentrations of allergen-specific IgG antibodies in dog sera.

An ELISA assay which uses horseradish peroxidase conjugated anti-canine IgG and polystyrene microtiter wells for detection of allergen-specific IgG in the serum of dogs is described. Individual allergen blanks were used to account for the variable nonspecific binding among various allergens, and the results observed in milliunits of absorbance were normalized using four reference sera. The coefficients of variation for the intraassay and interassay variability ranged from 1.34 to 12.50% and 4.62 to 9.77%, respectively. The relationship between ELISA results and serum concentrations of allergen-specific IgG was quantified. IgG antibodies with specificity for various allergens were found in the majority of non-atopic individuals and in all atopic subjects. Specific immunotherapy resulted in a rise in the serum concentration of allergen-specific IgG.