Marc Roger Couturier

Optimization of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis for Bacterial Identification

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.

Detection of Intestinal Protozoa in the Clinical Laboratory

ABSTRACT Despite recent advances in diagnostic technology, microscopic examination of stool specimens remains central to the diagnosis of most pathogenic intestinal protozoa. Microscopy is, however, labor-intensive and requires a skilled technologist. New, highly sensitive diagnostic methods have been developed for protozoa endemic to developed countries, including Giardia lamblia (syn. G. intestinalis/G. duodenalis) and Cryptosporidium spp., using technologies that, if expanded, could effectively complement or even replace microscopic approaches. To date, the scope of such novel technologies is limited and may not include common protozoa such as Dientamoeba fragilis, Entamoeba histolytica, or Cyclospora cayetanensis. This minireview describes canonical approaches for the detection of pathogenic intestinal protozoa, while highlighting recent developments and FDA-approved tools for clinical diagnosis of common intestinal protozoa.

Identification of HACEK clinical isolates by matrix-assisted laser desorption ionization-time of flight mass spectrometry

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry is a rapid and accurate tool for the identification of many microorganisms. We assessed this technology for the identification of 103 Haemophilus parainfluenzae, Aggregatibacter aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae (HACEK) clinical isolates and 20 Haemophilus influenzae clinical isolates. Ninety-three percent of HACEK organisms were identified correctly to the genus level using the Bruker database, and 100% were identified to the genus level using a custom database that included clinical isolates.

Leptotrichia Bacteremia in Patients Receiving High-Dose Chemotherapy

ABSTRACT Leptotrichia spp. are anaerobic, pencil-shaped, Gram-negative rods that are part of the normal oral and intestinal human flora. Although not typically considered pathogenic, invasive Leptotrichia infections have been reported in immunosuppressed patients. A perceived rise in the identification of Leptotrichia spp. at our institution prompted a retrospective evaluation of these infections. Laboratory and clinical records were reviewed to identify Leptotrichia culture-positive patients. Over a 5-year period, 68 Leptotrichia-positive specimens were identified. Of these, 21% (14/68) were identified in original samples submitted from 13 different patients at our institution, and the remainder (79% [54/68]) were unknown isolates referred from outside hospitals for molecular identification. All in-house Leptotrichia were identified from blood cultures. Only 64% (9/14) of these grew on solid media, and 5 were a part of polymicrobial bacteremias containing other enteric pathogens. All local patients were receiving chemotherapy and a majority received hematopoietic stem cell transplant (HSCT) (11/13). All had neutropenic fever with symptoms of mucositis and/or enteritis. Most of the HSCT patients (73% [8/11]) were autologous recipients hospitalized after recent high-dose chemotherapy for multiple myeloma. L. hongkongensis, a novel species, was found in the majority of myeloma cases (63% [5/8]). In conclusion, we suggest that Leptotrichia spp. may be an underappreciated cause of bacteremia, particularly in multiple myeloma patients receiving cytotoxic chemotherapy for autologous HSCT. In our cohort, these infections were associated with neutropenic fever from an enteric source, and most isolates remained sensitive to standard antibiotics.

Shiga-Toxigenic Escherichia coli Detection in Stool Samples Screened for Viral Gastroenteritis in Alberta, Canada

ABSTRACT Shiga-toxigenic Escherichia coli (STEC) is an important cause of diarrheal disease. The most notorious STEC serotype is O157:H7, which is associated with hemorrhagic colitis and hemolytic-uremic syndrome (HUS). As a result, this serotype is routinely screened for in clinical microbiology laboratories. With the bias toward the identification of the O157 serogroup in routine diagnostic processes, non-O157 STEC has been largely underrepresented in the epidemiology of STEC infections. This diagnostic bias is further complicated by the fact that many non-O157 STEC infections cause nonspecific gastroenteritis symptoms reminiscent of enteric viral infections. In this study, real-time PCR was used to amplify Shiga toxin genetic determinants (stx 1 and stx 2) from enriched stool samples that were initially submitted for the testing of enteric viruses in patients with suspected viral gastroenteritis between May and September of 2006, 2007, and 2008 (n = 2,702). Samples were submitted from the province of Alberta, Yukon, the Northwest Territories, and Nunavut, Canada. A total of 38 samples (1.4%) tested positive for Shiga toxin genes, and 15 isolates were cultured for further characterization. Several of the serotypes identified (O157:H7, O26:HNM, O26:H11, O103:H25, O121:H19, and O145:HNM) have been previously associated with outbreaks and HUS. This study outlines the importance of combining molecular methods with classical culture techniques to enhance the detection of emerging non-O157 as well as O157 serotypes in diarrheal stool samples. Furthermore, atypical diarrhea disease caused by non-O157 STEC can be routinely missed due to screening only for viral agents.

Evaluation of the FilmArray® Respiratory Panel for clinical use in a large children's hospital.

Respiratory pathogens are a leading cause of hospital admission and traditional detection methods are time consuming and insensitive. Multiplex molecular detection methods have recently been investigated in hope of replacing these traditional techniques with rapid panel‐based testing.

Economic Analysis of Rapid and Sensitive Polymerase Chain Reaction Testing in the Emergency Department for Influenza Infections in Children

Background: Rapid multiplex polymerase chain reaction (PCR) assays simultaneously detect several respiratory viral pathogens with high sensitivity. Maximizing detection of influenza at the point of care has the potential to reduce unnecessary antibiotic use, laboratory tests and hospitalizations. However, the cost-effectiveness of rapid multiplex PCR assays for influenza has not been compared with other diagnostic methods in children. Methods: For children presenting to the emergency department with influenza-like illness, we compared costs and outcomes using 4 different testing strategies for detection of influenza: (1) a rapid multiplex PCR platform (FilmArray); (2) traditional PCR; (3) direct-fluorescent antibody and (4) rapid antigen tests. Costs were assessed from the hospital perspective, and effectiveness was defined as quality-adjusted life years (QALYs). Input parameters were obtained from previous studies, and the model was run separately for children aged 3–36 months and 3–18 years. Results: Rapid multiplex PCR testing was the most effective testing strategy for children in both age groups. The incremental cost-effectiveness when compared with rapid antigen tests was

Comparison of Three Immunoassays for Detection of Antibodies to Strongyloides stercoralis

115,556 per QALY for children aged 3–36 months and from

Multiplexed Molecular Diagnostics for Respiratory, Gastrointestinal, and Central Nervous System Infections

228,000 per QALY for children aged 3–18 years. The cost-effectiveness of rapid multiplex PCR was sensitive to estimates for influenza prevalence, the proportion of patients treated with antivirals and the cost per test. Conclusions: Our model identifies scenarios in which identification of influenza in the emergency department using rapid multiplex PCR testing is a cost-effective strategy for infants and children 3 months through 18 years. Including detection of other respiratory viruses in the analysis would further improve cost-effectiveness.

Urine Antigen Tests for the Diagnosis of Respiratory Infections: Legionellosis, Histoplasmosis, Pneumococcal Pneumonia

ABSTRACT Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.