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.

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.

Staggered enforcement of infection control and prevention measures following four consecutive potential laboratory exposures to imported Brucella melitensis.

From 2015 until 2020, Brucella melitensis was isolated four times in our microbiology laboratory. All patients had travelled in endemic-areas. Immediately after the first occurrence, all laboratory staff were risk-stratified and preventive and protective measures were applied according to CDC guidelines. Nineteen workers were exposed and needed chemoprophylaxis and follow-up. At each subsequent occurrence, risk analysis was performed, and additional measures were implemented accordingly, leading to a progressive reduction of exposed staff members to none the fourth time. We describe here the additional measures that permitted this important exposure reduction.

Reactivation of Plasmodium infection during a treatment with infliximab: A case report.

We describe a symptomatic Plasmodium falciparum infection in a 29-year-old Guinean man receiving Infliximab for one year and without recent travel. The reactivation of submicroscopic malaria following the inhibition of TNF-alpha by infliximab is suspected.

[Campylobacter jejuni meningitis]. / Méningite à Campylobacter jejuni.

Cases of Campylobacter jejuni meningitis are extremely rare. In the literature, less than ten cases have been described so far, although Campylobacter spp is one of the most common pathogens causing gastroenteritis in the world. Some common stigmata can be found across these cases such as rupture of the blood-brain barrier, immunosuppression, as well as the tropism of Camplylobacter jejuni for neurological parenchyma. Campylobacter jejuni bacteremia is certainly underestimated because Campylobacter is a thermophilic bacterium and special conditions are required to isolate this organism in blood cultures. PCR is thus an interesting alternative technique for diagnosis. In our case, a patient with a history of resected astrocytoma, had undergone treatment with chemotherapy and radiotherapy because of anaplastic transformation. The patient was admitted with gastroenteritis and Campylobacter jejuni meningitis. The diagnosis was obtained initially on stool cultures and then by PCR of cerebrospinal fluid. The evolution was favorable with meropenem.

Les cas de méningite à Campylobacter jejuni restent extrêmement rares. Dans la littérature, on décrit moins de 10 cas à ce jour, alors que l'infection à Campylobacter est cependant l'une des causes les plus répandues de gastro-entérite dans le monde. Le point commun de tous ces cas de méningite rapportés semble être la fragilité de la barrière hémato-encéphalique et l'immunodépression, ainsi que le tropisme du Campylobacter jejuni pour les tissus neuronaux. La bactériémie à Campylobacter jejuni est par ailleurs sous-estimée car le germe est thermophilique et des conditions particulières sont nécessaires pour isoler cet organisme dans les hémocultures. La PCR est une alternative intéressante pour le diagnostic microbiologique. Dans le cas décrit, le patient présentait des antécédents d'astrocytome pariéto-temporal droit opéré, avec une transformation anaplasique ayant bénéficié de chimio- et radiothérapie concomitantes. Le patient a été admis avec une gastro-entérite compliquée d'une méningite à Campylobacter jejuni. Le diagnostic a été posé initialement sur la coproculture et ensuite par la PCR du liquide céphalo-rachidien. L'évolution a été favorable sous méropénem.

Enteropathogens in paediatric gastroenteritis: comparison of routine diagnostic and molecular methods.

OBJECTIVES: Studies of acute gastroenteritis (AGE) are hampered by the lack of routine diagnostic methods with good sensitivity and specificity. Molecular methods are increasingly used for clinical purposes, but the clinical significance of a positive result remains a challenge. In this study we aimed to compare results of routine diagnostic methods and molecular methods in symptomatic children and asymptomatic controls. METHODS: Patients presenting to the pediatric emergency departments of two university hospitals in Brussels with AGE were recruited prospectively from May 2015 to October 2016; asymptomatic controls were recruited from the same hospitals. Stool analyses were performed for all participants for common pathogenic bacteria (culture), virus (immunochromatography) and parasites (microscopy). Stools were also analysed with the Luminex Gastrointestinal Pathogen Panel, a multiplex-PCR for common enteropathogens. RESULTS: Stools from 178 patients and 165 controls were analysed. An enteropathogen was detected in 62.4% (111/178) of cases when combining the two methods (56.2% (100/178) by Luminex, 42.7% (76/178) with routine methods) and 29.1% (48/165) of controls (24.2% (40/165) by Luminex and 10.3% (17/165) by routine methods). Some pathogens were detected more often with Luminex than with routine methods, such as Salmonella (16.3% (29/178) with Luminex and 3.9% (7/178) with routine method, p < 0.05), whereas others identified by culture methods, such as Campylobacter, Shigella, Yersinia, were missed by Luminex. CONCLUSIONS: Molecular tools seem attractive methods, providing high positivity and a rapid turn-around time for the diagnosis of AGE. However, high rates of positivity in both cases and controls highlight the difficulty in interpreting results. Pathogens missed by Luminex but detected by culture methods raise more questions about the true clinical interest of the technique for our patients.

First case of Campylobacter rectus and Solobacterium moorei mixed bacteraemia successfully identified by MALDI TOF-MS.

Campylobacter rectus and Solobacterium moorei are anaerobic Gram-negative and Gram-positive rods, respectively, that are occasionally members of the human oral flora. Bacteraemia has rarely been reported. We present the first case of mixed C. rectus-S. moorei bacteraemia in an individual with diabetes and human immunodeficiency virus infection. Both bacteria were successfully identified by MALDI-TOF MS.

Relation between broiler and human Campylobacter jejuni strains isolated in Belgium from 2011 to 2013.

AIMS: This study inquires the relationship between Campylobacter jejuni isolated from broiler meat carcasses (n = 97) and human clinical samples (n = 72) in Belgium, from 2011 to 2013. METHODS AND RESULTS: The evaluation of the relation was based on the characteristics determined using multilocus sequence typing (MLST) alone and combined with flagellin gene A restriction fragment length polymorphism (flaA-RFLP) typing, antibiotic microbiological resistance profiling (AMRp), lipooligosaccharide class typing or virulence gene profiling (Vp). Clusters containing both human and broiler meat strains were more common when MLST was used alone, followed by MLST/flaA-RFLP and then by MLST/AMRp. More logical chronologically relations broiler-human were obtained for MLST/flaA-RFLP, then for MLST, and finally for MLST/AMRp: i.e. the isolates would first be detected in the broiler meat and at the same time or later in humans. CONCLUSIONS: In several cases, the C. jejuni strains isolated from the consumed broiler meat and from the campylobacteriosis case had the same profile, according to the used typing methods. The circulating Campylobacter strains appear to have remained the same from 2011 till 2013 in Belgium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study corroborates previously published data from Belgium that suggest a strong correlation between C. jejuni strains isolated from broiler meat and from campylobacteriosis patients.

MALDI-TOF MS contribution to the diagnosis of Campylobacter rectus multiple skull base and brain abscesses.

Campylobacter rectus is rarely associated with invasive infection. Both the isolation and the identification requirements of C. rectus are fastidious, probably contributing to an underestimation of its burden. We report the case of a 66-year-old man who developed several skull base and intracerebral abscesses after dental intervention. Campylobacter rectus was isolated from the brain biopsy. Within 45 minutes of reading the bacterial plate, the strain was accurately identified by MALDI-TOF MS. This rapid identification avoided the extra costs and delays present with 16S rRNA gene sequencing and allowed for a rapid confirmation of the adequacy of the empirical antibiotic treatment.

Antibiotic susceptibility profiles among Campylobacter isolates obtained from international travelers between 2007 and 2014.

Campylobacter infection is a common cause of diarrhea among international travelers. We studied antibiotic resistance patterns among Campylobacter isolates obtained from international travelers according to travel destination. Three collections of isolates obtained from international travelers between 2007 and 2014 (Institute of Tropical Medicine, the "Laboratoire Hospitalier Universitaire de Bruxelles "and the Belgian National Reference Centre for Campylobacter) were used. Isolates were tested for minimal inhibitory concentration (MIC) values (E-test macromethod) for fluoroquinolones, macrolides, tetracyclines, amoxicillin-clavulanic acid, and meropenem. Single isolates from 261 travelers were available; median (IQR) age was 25.4 (4-42) years, 85.8% were symptomatic (information for 224 patients available). Overall resistance to ciprofloxacin was 60.9%, ranging from 50.8% in Africa to 75.0% in Asia. Resistance to erythromycin was 4.6%, with the highest rate observed for Southern Asia (15.2%, seven isolates, six of them recovered from patients returning from India). A total of 126 isolates (48.3%) were resistant to tetracycline. No resistance to amoxicillin-clavulanic acid or meropenem was detected. Ciprofloxacin resistance tended to increase over time (53.9% in 2007 versus 72.2% in 2014), erythromycin resistance remained stable (median annual resistance 4.2%). Most (86.2%) ciprofloxacin-resistant isolates had MIC values ≥32 mg/l, and all erythromycin-resistant isolates had MIC values ≥256 mg/l. Co-resistance to ciprofloxacin and erythromycin was observed in 11 (4.2%) isolates, seven of which came from Southern Asia. Among all regions of travel, more than half of Campylobacter isolates were resistant to ciprofloxacin. Overall resistance to erythromycin was below 5% but reached 15.2% in Southern Asia.

Subtype determination of Blastocystis isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

The pathogenic role of the enteric parasite Blastocystis remains controversial. Recent studies have suggested that various subtypes (STs) found in human samples could be correlated to the presence or absence and variability of clinical manifestations, and that STs can differ with respect to drug sensitivity. Polymerase chain reaction (PCR) techniques used to determine these STs are expensive and are usually restricted to research laboratory settings. This study evaluates the potential application of the inexpensive matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) technique to discriminate Blastocystis STs. A database of parasitic protein signatures was constructed for five Blastocystis STs, and the reference spectra were challenged with those from 19 axenic cultures of ST1, ST2, ST3, ST4 and ST8 and those from nine xenic liquid cultures of ST3 and ST4. Samples from axenic cultures were prepared using standard formic acid extraction and direct deposition procedures. The reference spectra revealed five distinct spectral profiles, and the database library allowed for discrimination between all of the cultures with reliability indices ranging from 2.038 to greater than 2.8 when an extraction was performed. The direct deposition procedure resulted in greater variability in the discrimination and direct MALDI-TOF MS identification from xenic liquid cultures was effective in 3 out of 9 samples. MALDI-TOF MS proved to be an effective technology for efficiently discriminating Blastocystis STs in axenic cultures.

Feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) networking in university hospitals in Brussels.

The mutualisation of analytical platforms might be used to address rising healthcare costs. Our study aimed to evaluate the feasibility of networking a unique matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) system for common use in several university hospitals in Brussels, Belgium. During a one-month period, 1,055 successive bacterial isolates from the Brugmann University Hospital were identified on-site using conventional techniques; these same isolates were also identified using a MALDI-TOF MS system at the Porte de Hal Laboratory by sending target plates and identification projects via transportation and the INFECTIO_MALDI software (Infopartner, Nancy, France), respectively. The occurrence of transmission problems (<2 %) and human errors (<1 %) suggested that the system was sufficiently robust to be implemented in a network. With a median time-to-identification of 5 h and 11 min (78 min, min-max: 154-547), MALDI-TOF MS networking always provided a faster identification result than conventional techniques, except when chromogenic culture media and oxidase tests were used (p < 0.0001). However, the limited clinical benefits of the chromogenic culture media do not support their extra cost. Our financial analysis also suggested that MALDI-TOF MS networking could lead to substantial annual cost savings. MALDI-TOF MS networking presents many advantages, and few conventional techniques (optochin and oxidase tests) are required to ensure the same quality in patient care from the distant laboratory. Nevertheless, such networking should not be considered unless there is a reorganisation of workflow, efficient communication between teams, qualified technologists and a reliable IT department and helpdesk to manage potential connectivity problems.

Impact of rapid microbial identification directly from positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry on patient management.

For septic patients, delaying the initiation of antimicrobial therapy or choosing an inappropriate antibiotic can considerably worsen their prognosis. This study evaluated the impact of rapid microbial identification (RMI) from positive blood cultures on the management of patients with suspected sepsis. During a 6-month period, RMI by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed for all new episodes of bacteraemia. For each patient, the infectious disease specialist was contacted and questioned about his therapeutic decisions made based on the Gram staining and the RMI. This information was collected to evaluate the number of RMIs that led to a therapeutic change or to a modification of the patient's general management (e.g. fast removal of infected catheters). During the study period, 277 new episodes of bacteraemia were recorded. In 71.12% of the cases, MALDI-TOF MS resulted in a successful RMI (197/277). For adult and paediatric patients, 13.38% (21/157) and 2.50% (1/40) of the RMIs, respectively, resulted in modification of the treatment regimen, according to the survey. In many other cases, the MALDI-TOF MS was a helpful tool for infectious disease specialists because it confirmed suspected cases of contamination, especially in the paediatric population (15/40 RMIs, 37.50%), or suggested complementary diagnostic testing. This study emphasizes the benefits of RMI from positive blood cultures. Although the use of this technique represents an extra cost for the laboratory, RMI using MALDI-TOF MS has been implemented in our daily practice.

Comparison of an in-house method and the commercial Sepsityper™ kit for bacterial identification directly from positive blood culture broths by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry.

The identification of bacteria directly from positive blood cultures using matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a new challenge to microbiologists. However, the protocols previously described are often difficult to implement in routine and comparisons are not always possible due to the variability of interpretative criteria. This study evaluated the analytical and practical performances of an in-house (IH) method, adapted from previous protocols, and the Sepsityper™ kit (Bruker Daltonics, Bremen, Germany). Positive blood cultures from 63 different patients were prospectively evaluated by both methods. To enhance the sensitivity of these methods, lowered cut-offs were assessed and validated on 66 additional samples. The IH method produced 86.4% and 73.7% correct genus and species identifications, respectively, when using the lowered cut-offs of 1.4 and 1.6 for correct genus and species identifications. The Sepsityper™ kit showed similar results (78.0% and 68.4% correct genus and species identification, respectively). However, the IH method is ten-fold less expensive than the commercial option (0.72 vs. 7.45 /analysis) and its turnaround time is approximately 20 min versus the nearly 40 min required for the Sepsityper™ kit, which includes an extraction step. Finally, the IH method was introduced twice-daily in our routine practice.

Accuracy of the API Campy system, the Vitek 2 Neisseria-Haemophilus card and matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of Campylobacter and related organisms.

Biochemical identification of Campylobacter and related organisms is not always specific, and may lead to diagnostic errors. The API Campy, the Vitek 2 system and matrix-assisted desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) are commercially available methods that are routinely used for the identification of these microorganisms. In the present study, we used 224 clinical isolates and ten reference strains previously identified by multiple PCR assays, whole cell protein profiling and either DNA-DNA hybridization or sequencing analysis to compare the reliability of these three methods for the identification of Campylobacter and related pathogens. The API Campy accurately identified 94.4% of Campylobacter jejuni ssp. jejuni and 73.8% of Campylobacter coli, but failed to correctly identify 52.3% of other Epsilobacteria. The Vitek 2 Neisseria-Haemophilus card correctly identified most C. jejuni ssp.jejuni (89.6%) and C. coli (87.7%) strains, which account for the majority of campylobacterioses reported in humans, but it failed in the identification of all of the other species. Despite a good identification rate for both C. jejuni ssp. jejuni and C. coli, both methods showed poor sensitivity in the identification of related organisms, and additional tests were frequently needed. In contrast to API Campy and Vitek, MALDI-TOF MS correctly identified 100% of C. coli and C. jejuni strains tested. With an overall sensitivity of 98.3% and a short response time, this technology appears to be a reliable and promising method for the routine identification of Campylobacter and other Epsilobacteria.