Delphine Martiny

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.

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 patients 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.

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.

Identification of the Trichophyton mentagrophytes complex species using MALDI-TOF mass spectrometry

Dermatophytes are fungi capable of invading keratinized tissues and are responsible for the most common fungal infection worldwide: dermatophytosis. Identification of these organisms to the species level is often necessary for the correct treatment of these infections, and is always recommended from an epidemiological point of view. Since the identification of dermatophytes is sometimes problematic, we assessed whether Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) could provide a useful tool to identify dermatophytes of the Trichophyton mentagrophytes complex. A reference database was constructed with 17 strains of six different species belonging to this complex. A total of 54 dermatophyte strains of the Belgian co-ordinated collections of micro-organisms, Scientific Institute of Public Health, Brussels, Belgium (BCCM™/IHEM) collection were used to challenge this database; 89% of the tested strains (not used as reference strains in the database) could readily be identified. When incorrect identifications were encountered, the confusion was always between phylogenetically closely related taxa which indicates that observations made by MALDI-TOF MS correlate with phylogenetic data. To assess this observation, a dendrogram outlining the similarities between the obtained spectra was constructed. Strikingly, the relationships found in this dendrogram were highly similar to the ones observed in the phylogenetic tree recently reported by Beguin and co-workers. In conclusion, MALDI-TOF MS is a fast and reliable tool for the identification of dermatophytes, since it can even discriminate between the closely related species of the T. mentagrophytes complex. Moreover, our data indicate that the data obtained by MALDI-TOF MS correlate with phylogenetic data.

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.

Optimization of Campylobacter growth conditions for further identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Growth conditions - including growth medium and incubation temperature - may influence the identification of Campylobacter by MALDI-TOF MS. For each bacterial species, medical microbiologists should be aware of such potential influences on spectral data before analyzing and interpreting MALDI-TOF MS results.

Same-Day Subtyping of Campylobacter jejuni and C. coli Isolates by Use of Multiplex Ligation-Dependent Probe Amplification–Binary Typing

ABSTRACT Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping Campylobacter jejuni and Campylobacter coli. It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.

Clinical bacteriology in low-resource settings: today's solutions

Low-resource settings are disproportionately burdened by infectious diseases and antimicrobial resistance. Good quality clinical bacteriology through a well functioning reference laboratory network is necessary for effective resistance control, but low-resource settings face infrastructural, technical, and behavioural challenges in the implementation of clinical bacteriology. In this Personal View, we explore what constitutes successful implementation of clinical bacteriology in low-resource settings and describe a framework for implementation that is suitable for general referral hospitals in low-income and middle-income countries with a moderate infrastructure. Most microbiological techniques and equipment are not developed for the specific needs of such settings. Pending the arrival of a new generation diagnostics for these settings, we suggest focus on improving, adapting, and implementing conventional, culture-based techniques. Priorities in low-resource settings include harmonised, quality assured, and tropicalised equipment, consumables, and techniques, and rationalised bacterial identification and testing for antimicrobial resistance. Diagnostics should be integrated into clinical care and patient management; clinically relevant specimens must be appropriately selected and prioritised. Open-access training materials and information management tools should be developed. Also important is the need for onsite validation and field adoption of diagnostics in low-resource settings, with considerable shortening of the time between development and implementation of diagnostics. We argue that the implementation of clinical bacteriology in low-resource settings improves patient management, provides valuable surveillance for local antibiotic treatment guidelines and national policies, and supports containment of antimicrobial resistance and the prevention and control of hospital-acquired infections.

Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age

&NA; Thermotolerant Campylobacter spp. are a major cause of foodborne gastrointestinal infections worldwide. The linkage of human campylobacteriosis and poultry has been widely described. In this study we aimed to investigate the prevalence, antimicrobial resistance and genetic diversity of C. coli and C. jejuni in broilers from Ecuador. Caecal content from 379 randomly selected broiler batches originating from 115 farms were collected from 6 slaughterhouses located in the province of Pichincha during 1 year. Microbiological isolation was performed by direct plating on mCCDA agar. Identification of Campylobacter species was done by PCR. Minimum inhibitory concentration (MIC) values for gentamicin, ciprofloxacin, nalidixic acid, tetracycline, streptomycin, and erythromycin were obtained. Genetic variation was assessed by RFLP‐flaA typing and Multilocus Sequence Typing (MLST) of selected isolates. Prevalence at batch level was 64.1%. Of the positive batches 68.7% were positive for C. coli, 18.9% for C. jejuni, and 12.4% for C. coli and C. jejuni. Resistance rates above 67% were shown for tetracycline, ciprofloxacin, and nalidixic acid. The resistance pattern tetracycline, ciprofloxin, and nalidixic acid was the dominant one in both Campylobacter species. RFLP‐flaA typing analysis showed that C. coli and C. jejuni strains belonged to 38 and 26 profiles respectively. On the other hand MLST typing revealed that C. coli except one strain belonged to CC‐828, while C. jejuni except 2 strains belonged to 12 assigned clonal complexes (CCs). Furthermore 4 new sequence types (STs) for both species were described, whereby 2 new STs for C. coli were based on new allele sequences. Further research is necessary to estimate the impact of the slaughter of Campylobacter positive broiler batches on the contamination level of carcasses in slaughterhouses and at retail in Ecuador.

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.