Sonia Chatellier

Biomedical Mass Spectrometry in Today's and Tomorrow's Clinical Microbiology Laboratories

ABSTRACT Clinical microbiology is a conservative laboratory exercise where base technologies introduced in the 19th century remained essentially unaltered. High-tech mass spectrometry (MS) has changed that. Within a few years following its adaptation to microbiological diagnostics, MS has been introduced, embraced, and broadly accepted by clinical microbiology laboratories throughout the world as an innovative tool for definitive bacterial species identification. Herein, we review the current state of the art with respect to this exciting new technology and discuss potential future applications.

Comparison of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry and Molecular Biology Techniques for Identification of Culicoides (Diptera: Ceratopogonidae) Biting Midges in Senegal

ABSTRACT Biting midges of the genus Culicoides are implicated as vectors for a wide variety of pathogens. The morphological identification of these arthropods may be difficult because of a lack of detailed investigation of taxonomy for this species in Africa. However, matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) profiling is efficient for arthropod identification at the species level. This study established a spectrum database of Culicoides spp. from Senegal using MALDI-TOF. Identification of Culicoides insects to the species level before mass spectrometry was performed on the basis of morphological characters. MALDI-TOF MS reference spectra were determined for 437 field-caught Culicoides of 10 species. The protein profiles of all tested Culicoides revealed several peaks with mass ranges of 2 to 20 kDa. In a validation study, 72 Culicoides specimens in the target species were correctly identified at the species level with a similarity of 95 to 99.9%. Four Culicoides protein profiles were misidentified. Nevertheless, six SuperSpectra (C. imicola, C. enderleini, C. oxystoma, C. kingi, C. magnus, and C. fulvithorax) were created. Abdomens of midges were used to amplify and sequence a portion of the mitochondrial cytochrome oxidase I gene (COI). The results obtained using the MALDI-TOF MS method were consistent with the morphological identification and similar to the genetic identification. Protein profiling using MALDI-TOF is an efficient approach for the identification of Culicoides spp., and it is economically advantageous for approaches that require detailed and quantitative information of vector species that are collected in field. The database of African Culicoides MS spectra created is the first database in Africa. The COI sequences of five Culicoides species that were previously noncharacterized using molecular methods were deposited in GenBank.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

The Ongoing Revolution of MALDI-TOF Mass Spectrometry for Microbiology Reaches Tropical Africa

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.

A Novel Solid Medium for Culturing Mycobacterium tuberculosis Isolates from Clinical Specimens

ABSTRACT The laboratory diagnosis of tuberculosis usually relies on culture-based isolation of the causative Mycobacterium tuberculosis bacteria. We developed and evaluated the performance of MOD9, a new blood-free derivative of the MOD4 solid medium on which we previously reported for the isolation and culture of mycobacteria. First, inoculation of Lowenstein-Jensen medium with 21 M. tuberculosis isolates at 105, 103, and 10 CFU yielded colonies in 5.7 ± 1.5 days, 7.6 ± 0.8 days, and 10.8 ± 1.7 days versus 1.5 ± 0.4 days, 3.5 ± 0.6 days, and 4.9 ± 1 days in MOD9 (P < 0.05, Students t test). Further, the time to detectable growth of M. tuberculosis was measured on MOD9 and Lowenstein-Jensen media after duplicate inoculation of 250 clinical specimens decontaminated with 0.7% chlorhexidine. The contamination rate was 1.6% (4/250) on MOD9 versus 4.4% (11/250) on Lowenstein-Jensen medium (P = 0.11, Fishers exact test). Chlorhexidine-MOD9 yielded 38/250 (15.2%) isolates versus 32/250 (12.8%) isolates for the chlorhexidine-LJ (P = 0.5195, Fishers exact test). All together, eight M. tuberculosis isolates were cultured solely from chlorhexidine-MOD9, and two M. tuberculosis isolates were cultured solely from chlorhexidine-LJ. The time to detection was 9.8 ± 3.9 (range, 5 to 18) days for chlorhexidine-MOD9 versus 17.4 ± 5.9 (range, 10 to 35) days for chlorhexidine-LJ (P < 0.05, Students t test). These data indicate the superiority of the MOD9 medium over the standard LJ medium following chlorhexidine decontamination for the recovery of M. tuberculosis.

Challenges in the culture-independent analysis of oral and respiratory samples from intubated patients

The spread of microorganisms in hospitals is an important public health threat, and yet few studies have assessed how human microbial communities (microbiota) evolve in the hospital setting. Studies conducted so far have mainly focused on a limited number of bacterial species, mostly pathogenic ones and primarily during outbreaks. We explored the bacterial community diversity of the microbiota from oral and respiratory samples of intubated patients hospitalized in the intensive care unit and we discuss the technical challenges that may arise while using culture-independent approaches to study these types of samples.

Performance of solid and liquid culture media for the detection of Mycobacterium tuberculosis in clinical materials: meta-analysis of recent studies

Tuberculosis (TB) is a universal disease: it occurs all over the globe, albeit with hugely varying regional incidence. Especially in Asian and African countries, the incidence and prevalence may be extremely high. The European Journal of Clinical Microbiology and Infectious Diseases published seven papers on TB in 2013, representing 3.3 % (7/211) of its annual production. Four of the papers concerned mechanistic studies on clinical patient management, cost-effectiveness of antibiotic treatment, importance of microbiota and host genetic polymorphisms, respectively. The other three papers concerned molecular diagnostics, so, overall, hardly any attention was paid to the more classical modes of diagnosing TB. And this clearly contrasts with international needs, since classical diagnostics is still very high on the agenda in those countries where TB testing is most needed. In their 2013 Global Tuberculosis Report, the World Health Organization (WHO) claimed that culture is still the reference method for the detection of Mycobacterium tuberculosis (Mtb), with smear staining and microscopy being considered as a method of “added value”. Hence, a wide array of culture systems has been developed by a large multitude of researchers and companies. Some of these are semi-automated, but most require manual intervention. Lowenstein–Jensen medium (LJ) is frequently considered the key mycobacterial growth medium when comparative studies for the verification and validation of new diagnostic tests are performed. Even the validation of liquid media in combination with semi-automated culture systems is usually performed in comparison with LJ-based cultivation as the diagnostic Gold Standard. We have set out to investigate the quality of some of the culture media proposed for the detection of Mtb in order to verify whether such media really are the appropriate Gold Standard tools for the validation of novel TB diagnostics. We performed a literature search (PubMed dd 13 August 2013) for the period between January 2008 and August 2013. In this way, 71 relevant articles were identified, of which the 19 most complete studies were selected for more detailed analysis. We collected the number of specimens tested, the culture medium used and identified, where possible, the manufacturer of the media. As such, six different producers of culture media for Mtb detection were identified. When microscopic observation of drug susceptibility (MODS) was performed, we included these data as well. Overall, the highest sensitivities and negative predictive values were shown by the BACTEC MGIT 960 System (Becton-Dickinson, Sparks, MD, USA), either when used alone or in combination with LJ. The latter combination provided the best sensitivity and also scored particularly well in the detection of non-tuberculous mycobacteria. The highest specificity and positive predictive values were shown by the LJ method itself, which obviously produced no falsepositives. So, MGIT is the top performer with regard to sensitivity, while LJ sets the current standard for specificity. This is well in line with international findings and recommendations. As can be seen in Table 1, liquid media show times to positivity that are about half of those for solid media (about 14.2 days for MGIT versus 28 days for LJ). However, and this is important, afar-based media from various suppliers show differences in performance, with time to positivity averages ranging from 22.5 to 32 days. Assuming that there were no systematic differences in the bacterial loads of the samples F. Rageade :N. Picot :A. Blanc-Michaud : S. Chatellier : C. Mirande : E. Fortin :A. van Belkum (*) bioMerieux SA, Chemin de l’Orme, 69280 Marcy l’Etoile, France e-mail: alex.vanbelkum@biomerieux.com

Identification of mycobacterium spp. and nocardia spp. from solid and liquid cultures by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Identification of microorganisms by MALDI-TOF MS has been widely accepted in clinical microbiology. However, for Mycobacterium spp. and Nocardia spp. such identification has not yet reached the optimal level of routine testing. Here we describe the development of an identification tool for 49 and 15 species of Mycobacterium spp. and Nocardia spp., respectively. During database construction, a number of ambiguous reference identifications were revealed and corrected via molecular analyses. Eventually, more than 2000 individual mass spectra acquired from 494 strains were included in a reference database and subjected to bio-statistical analyses. This led to correct species identification and correct combination of species into several complexes or groups, such as the Mycobacterium tuberculosis complex. With the Advanced Spectrum Classifier algorithm, class-specific bin weights were determined and tested by cross-validation experiments with good results. When challenged with independent isolates, overall identification performance was 90% for identification of Mycobacterium spp. and 88% for Nocardia spp. However, for a number of Mycobacterium sp. isolates, no identification could be achieved and in most cases, this could be attributed to the production of polymers that masked the species-specific protein peak patterns. For the species where >20 isolates were tested, correct identification reached 95% or higher. With the current spectral database, the identification of Mycobacterium spp. and Nocardia spp. by MALDI-TOF MS can be performed in routine clinical diagnostics although in some complicated cases verification by sequencing remains mandatory.

A Chlorhexidine- Agar Plate Culture Medium Protocol to Complement Standard Broth Culture of Mycobacterium tuberculosis.

The culture of Mycobacterium tuberculosis using parallel inoculation of a solid culture medium and a liquid broth provides the gold standard for the diagnosis of tuberculosis. Here, we evaluated a chlorhexidine decontamination-MOD9 solid medium protocol versus the standard NALC-NaOH-Bactec 960 MGIT protocol for the diagnosis of pulmonary tuberculosis by culture. Three-hundred clinical specimens comprising 193 sputa, 30 bronchial aspirates, 10 broncho-alveolar lavages, 47 stools, and 20 urines were prospectively submitted for the routine diagnosis of tuberculosis. The contamination rates were 5/300 (1.7%) using the MOD9 protocol and 17/300 (5.7%) with the Bactec protocol, respectively (P < 0.05, Fisher exact test). Of a total of 50 Mycobacterium isolates (48 M. tuberculosis and two Mycobacterium abscessus) were cultured. Out of these 50, 48 (96%) isolates were found using the MOD9 protocol versus 35 (70%) when using the Bactec protocol (P < 0.05, Fisher exact test). The time to positivity was 10.1 ± 3.9 days versus 14.7 ± 7.3 days, respectively, (P < 0.05, Student’s t-test). These data confirmed the usefulness of parallel inoculation of a solid culture medium with broth for the recovery of M. tuberculosis in agreement with current recommendations. More specifically, chlorhexidine decontamination and inoculation of the MOD9 solid medium could be proposed to complement the standard Bactec 960 MGIT broth protocol.

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.