Abdessalam Cherkaoui

Kingella Kingae osteoarticular infections in young children: clinical features and contribution of a new specific real-time PCR assay to the diagnosis

Backgroung Kingella kingae is an emerging pathogen that may be recognized as the most common bacteria responsible for osteoarticular infections (OAI) in young children. However, its diagnosis remains a challenge and thus little evoked in infants, because K. kingae is a difficult germ to isolate on solid medium, and clinical signs are often mild. The main objective of this prospective study is to describe the clinical, biologic, and radiologic features of children with OAI caused by K. kingae. In addition, we describe the usage of a new specific real-time PCR assay in children under 4 years admitted for OAI with a probe that detects 2 independent gene targets from the K. kingae RTX toxin. Patients and Methods All children less than 4 years admitted in our institution between January 2007 and November 2009 for suspected OAI were enrolled in this prospective study (43 cases). Age, gender, clinical signs, duration of symptoms, bone or joint involved, imaging studies, and laboratory data, including bacterial investigations, full blood count, erythrocyte sedimentation rate, and serum C-reactive protein were collected for analysis. Results Identification of the microorganism was possible for 28 cases (65.1%) yielding K. kingae in 23 cases (82.1%). Mean age of children with K. kingae OAI was 19.6 months. Less than 15% of these patients were febrile during the admission, but 46% of them presented a history of fever-peak superior to 38.5°C before admission. Thirty-nine percent of the children with K. kingae OAI had normal C-reactive protein; WBC was elevated in only 2 cases, whereas 21 patients had abnormal erythrocyte sedimentation rate, and 13 abnormal platelet counts. Direct Gram staining and classical isolation methods were negative for all cases subsequently detected as K. kingae OAI by specific real-time PCR. Conclusion This study confirms that K. kingae is the major bacterial cause of OAI in children less than 4 years. The real-time PCR assay, specific to the K. kingae RTX toxin, provides interesting diagnostic performance when implemented in the routine microbiologic laboratory. Needless to say, a bigger cohort is required to adequately study this new qPCR assay, but the results so far seem promising. The most important additional finding is the mild-to-moderate clinical, radiologic, and biologic inflammatory response to K. kingae infection with the result that these children present few criteria evocative of OAI. Level of Evidence II

Use of PCR Coupled with Electrospray Ionization Mass Spectrometry for Rapid Identification of Bacterial and Yeast Bloodstream Pathogens from Blood Culture Bottles

ABSTRACT Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the methods clinical utility at this time.

Comparative analysis of PCR-electrospray ionization/mass spectrometry (MS) and MALDI-TOF/MS for the identification of bacteria and yeast from positive blood culture bottles

BACKGROUND Emerging technologies for rapid identification of microbes demonstrate a shift from traditional biochemical and molecular testing algorithms toward methods using mass spectrometry (MS) for the semiquantitative analysis of microbial proteins and genetic elements. This study was performed to assess the diagnostic accuracy of 2 such technologies, PCR–electrospray ionization (ESI)/MS and MALDI-TOF/MS, with respect to phenotypic and biochemical profiling as a reference standard method. A positive challenge set of blood culture bottles was used to compare PCR-ESI/MS and MALDI-TOF/MS performance on a matched set of samples. METHODS We performed characterization of bloodstream infections from blood cultures using the Ibis T5000 PCR-ESI/MS and the Bruker MALDI Biotyper 2.0 (MALDI-TOF/MS) platforms for microbial identification. Diagnostic accuracy was determined by independent comparison of each method to phenotypic and biochemical characterization with Vitek2 analysis as the reference standard identification. RESULTS The diagnostic accuracy, represented as positive agreement, at the genus level was 0.965 (0.930–0.984) for PCR-ESI/MS and 0.969 (0.935–0.987) for MALDI-TOF/MS, and at the species level was 0.952 (0.912–0.974) with PCR-ESI/MS and 0.943 (0.902–0.968) for MALDI-TOF/MS. No statistically significant difference was found between PCR-ESI/MS and MALDI-TOF/MS in the ability to rapidly identify microorganisms isolated from blood culture. CONCLUSIONS Our results demonstrate that PCR-ESI/MS and MALDI-TOF/MS are equivalent in their ability to characterize bloodstream infections with respect to the reference standard, and highlight key differences in the methods that allow for each method to have a unique niche as a tool for rapid identification of microbes in blood cultures.

Molecular analysis of NDM-1-producing enterobacterial isolates from Geneva, Switzerland

OBJECTIVES To analyse the mechanisms responsible for decreased susceptibility or resistance to carbapenems in several enterobacterial isolates recovered in 2009-10 in Geneva University Hospitals, Switzerland. METHODS PCR and sequencing were used to identify β-lactamases, 16S RNA methylases and plasmid-mediated quinolone resistance genes. The transferable properties of the plasmids were analysed, as well as their plasmid type. The strains were typed by multilocus sequence typing. RESULTS Three patients were found to be positive for NDM-1-producing enterobacterial isolates (one with Escherichia coli and Klebsiella pneumoniae, one with K. pneumoniae only and one with Proteus mirabilis), where NDM-1 stands for New Delhi metallo-β-lactamase-1. The bla(NDM-1) carbapenemase gene was detected in all isolates in addition to genes encoding narrow-spectrum β-lactamases (TEM-1, SHV-11, OXA-1, OXA-9 and OXA-10), extended-spectrum β-lactamases (CTX-M-15, CMY-16 and CMY-30), ArmA and quinolone resistance determinants (Qnr). The bla(NDM-1) gene was located on conjugative IncA/C- or IncF-type plasmids. Upstream of the bla(NDM-1) gene, part of ISAba125, previously identified in NDM-1-negative Acinetobacter baumannii, was found. Downstream of the bla(NDM-1) gene, variable sequences were found. CONCLUSIONS This work constitutes the first identification of NDM-1 producers in Switzerland. Interestingly, patients from whom these NDM-1-producing isolates were recovered had a link with the Indian subcontinent or the Balkans.

Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay.

Kingella kingae is an emerging pathogen that is recognized as a causative agent of septic arthritis and osteomyelitis, primarily in infants and children. The bacterium is best detected by rapid inoculation in blood culture systems or by real-time PCR assays. Pathogenesis of the agent was linked recently to the production of a potent cytotoxin, known as RTX, which is toxic to a variety of human cell types. The locus encoding the RTX toxin is thought to be a putative virulence factor, and is, apparently, essential for inducing cytotoxic effects on respiratory epithelial, synovial and macrophage-like cells. Herein, we describe a novel real-time PCR assay that targets the RTX toxin gene and illustrate its use in two clinical cases. The assay exhibited a sensitivity of 30 c.f.u., which is 10-fold more sensitive than a previously published semi-nested broad-range 16S rRNA gene PCR, and showed no cross-reactivity with several related species and common osteoarticular pathogens.

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Identification of Beta-Hemolytic Streptococci

ABSTRACT This study was undertaken to evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the rapid identification of beta-hemolytic streptococci. We compared Bruker Biotyper 2.0 with Vitek2 coupled to the agglutination test. MALDI-TOF MS analysis of 386 beta-hemolytic streptococcal isolates yielded high-confidence identification to the species level for all 386 isolates. The Vitek2 gave high-confidence identification to the species level for 88% of Streptococcus agalactiae isolates (n = 269/306), 92% of Streptococcus pyogenes isolates (n = 48/52), and 39% of isolates of Streptococcus dysgalactiae serogroups C and G (n = 11/28).

Detection of Kingella kingae Osteoarticular Infections in Children by Oropharyngeal Swab PCR

OBJECTIVE: The purpose of this study was to investigate if oropharyngeal swab polymerase chain reaction (PCR) could predict osteoarticular infection (OAI) due to Kingella kingae in young children. METHODS: One hundred twenty-three consecutive children aged 6 to 48 months presenting with atraumatic osteoarticular complaints were prospectively studied. All had a clinical evaluation, imaging, and blood samples. Blood and oropharyngeal specimens were tested with a PCR assay specific for K kingae. OAI was defined as bone, joint, or blood detection of pathogenic bacteria, or MRI consistent with infection in the absence of positive microbiology. K kingae OAI was defined by blood, bone, or synovial fluid positivity for the organism by culture or PCR. RESULTS: Forty children met the OAI case definition; 30 had K kingae OAI, 1 had another organism, and 9 had no microbiologic diagnosis. All 30 oropharyngeal swabs from the K kingae case patients and 8 swabs from the 84 patients without OAI or with OAI caused by another organism were positive. The sensitivity and specificity of the oropharyngeal swab PCR assay for K kingae were 100% and 90.5%, respectively. CONCLUSIONS: Detection of K kingae DNA in oropharyngeal swabs of children with clinical findings of OAI is predictive of K kingae OAI. If these findings are replicated in other settings, detection of K kingae by oropharyngeal swab PCR could improve the recognition of OAI.

Differentiating Osteoarticular Infections Caused By kingella Kingae From Those Due to Typical Pathogens in Young Children

Osteoarticular infections caused by Kingella kingae are characterized by mild-to-moderate clinical and biologic inflammatory signs that are different from those caused by Gram-positive cocci. A combined score was built to find the best model to predict K. kingae osteoarticular infections by using the following 4 variables: body temperature <38°C, serum C-reactive protein <55 mg/L, white blood cell count <14,000/mm3, and band forms <150/mm3.

Small risk of osteoarticular infections in children with asymptomatic oropharyngeal carriage of Kingella kingae.

The aim of this study was to evaluate the absolute risk for children younger than 4 years of age with asymptomatic oropharyngeal carriage of Kingella kingae to sustain an osteoarticular infection. The rate of K. kingae carriage in the oropharyngeal mucosa was 9% among healthy children, and the risk for an asymptomatic carrier to develop an osteoarticular infection due to K. Kingae was estimated to be lower than 1%.

Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology

Broad-range PCR followed by sequencing identifies bacterial pathogens, even in challenging settings such as patients receiving antibiotics or infected with fastidious or non-cultivable organisms. The major problem with broad-range PCR is the risk of sample contamination. Risk is present at every step of the procedure, starting from sample collection. Contaminating bacterial DNA may be present not only in laboratory reagents but also at the surface of plastic consumables and containers used for specimen drawing and transport to the diagnostic laboratory. Contaminating DNA is amplified efficiently, leading to false-positive results. Thus, high specificity depends on eliminating such spurious targets, an awkward problem given the abundance of such targets and a highly sensitive method that detects very small numbers of molecules. Several investigators have reported strategies for eliminating the amplification of contaminating DNA sequences. So far, none of these methods has been entirely effective and reproducible. Here we describe a method that uses Exonuclease III (ExoIII) to disable contaminating sequences from acting as templates, while maintaining the high sensitivity of PCR for pathogen DNA. We use this assay in 144 clinical specimens from normally sterile sites, identifying pathogens from 24 (17%). Conventional methods identified pathogens in only four of these specimens, all of which were positive for the same pathogen by PCR. Compared with conventional methods, broad-range PCR with ExoIII pre-treatment of reagents substantially improves the diagnostic yield of bacterial pathogen identification from normally sterile sites.