Oropharyngeal Kingella kingae carriage in children: characteristics and correlation with osteoarticular infections.

BACKGROUND: The aim of this study was to investigate changes in oropharyngeal K. kingae carriage during the first 4 y of life, including seasonal variation and comparison of asymptomatic carriage with cases of invasive osteoarticular infections (OAI). METHODS: Oropharyngeal bacterial K. kingae carriage was screened in 744 healthy children aged 7-48 mo between January 2009 and December 2012. Oropharyngeal swabs were analyzed by rt-PCR targeting the DNA of K. kingae RTX toxin, epidemiological characteristics of asymptomatic carriers and OAI case patients were recorded. RESULTS: The carriage prevalence showed no significant difference between age groups or seasons. Compared with asymptomatic carriers, OAI cases were more likely to be aged from 7 to 12 mo (OR = 2.5; 95% CI (1.2-5.0)) and 13-24 mo (OR = 2.2; 95% CI (1.2-3.9)), and less likely over 36 mo (OR = 0.2; 95% CI (0.1-0.7)). Fewer OAI cases were identified in spring compared to asymptomatic carriers (OR = 0.3; 95% CI (0.1-0.7)), while more were detected in autumn (OR = 2.5; 95% CI (1.4-4.4)). CONCLUSION: Although oropharyngeal K. kingae colonization is a prerequisite for further invasive infection, this epidemiological study emphasizes that the carriage rate variations do not correlate with the variations of OAI incidence by gender, season, or age group.

Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry methods with conventional phenotypic identification for routine identification of bacteria to the species level.

Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.

Effect of levofloxacin on the viability of intracellular Chlamydia pneumoniae and modulation of proinflammatory cytokine production by human monocytes.

Although antibiotics are known to affect the intracellular growth of Chlamydia pneumoniae in acute infections, their efficacy in therapy for chronic infections, including atherosclerosis, remains debatable. Human monocyte-derived macrophages (MDM) obtained from monocytes of healthy donors were infected with C. pneumoniae AR-39 and treated with levofloxacin (8 microg/mL) immediately after infection (0 hours) or 24 hours after infection. Levofloxacin treatment at 24 hours, but not at 0 hours, resulted in a significant decrease in the number of C. pneumoniae inclusions within the MDM (p < 0.05). Also decreased were concentrations of proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and IL-8 in the extracellular medium (p < 0.01). Viable counts in titrations remained similar to those in untreated controls. In summary, levofloxacin administered to MDM at serum-attainable levels 24 hours after C. pneumoniae infection significantly decreased inclusion counts and proinflammatory cytokine production, but did not eliminate the C. pneumoniae infection.

Fulminant atypical Cryptococcus neoformans pneumonia confirmed by PLEX-ID.

Use of the PLEX-ID system can lead to a rapid molecular diagnosis in microbiology. To illustrate the clinical implications of this new diagnostic tool, we present the case of a 46-year-old patient admitted with severe respiratory failure and septic shock. Cryptococcal pneumonia was diagnosed by Fungi-Fluor™ staining of the bronchoalveolar lavage (BAL) and the patient tested positive for HIV. Unfortunately, he died 12h after admission despite intensive care support and treatment with broad-spectrum antibiotics, amphotericin B, and flucytosine. Retrospective use of the PLEX-ID on the BAL, bronchial aspirate, and blood yielded Cryptococcus neoformans in all fluids tested. Rapid molecular diagnosis with PLEX-ID, especially when performed on the blood of septic patients, may reduce the time to adequate treatment and limit the number of diagnostic procedures needed.

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.

Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications.

We evaluated the robustness of loop-mediated isothermal amplification (LAMP) of DNA for bacterial diagnostic applications. Salmonella enterica serovar Typhi was used as the target organism and compared with a real-time quantitative PCR (qPCR) for testing assay performance and reproducibly, as well as the impact of pH and temperature stability. This isothermal amplification method appeared to be particularly robust across 2 pH units (7.3-9.3) and temperature values (57-67 °C). The detection limit was comparable to that observed using optimized home-brew qPCR assays. The specificity of the amplification reaction remained high even at temperatures markedly different from the optimal one. Exposing reagents to the ambient temperature during the preparation of the reaction mixture as well as prolonging times for preparing the amplification reaction did not yield false-positive results. LAMP remained sensitive and specific despite the addition of untreated biological fluids such as stool or urine that commonly inhibit PCR amplification. Whereas the detection of microorganisms from whole blood or a blood-culture medium typically requires extensive sample purification and removal of inhibitors, LAMP amplification remained more sensitive than conventional qPCR when omitting such preparatory steps. Our results demonstrate that LAMP is not only easy to use, but is also a very robust, innovative and powerful molecular diagnostic method for both industrialized and developing countries.

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.

Levofloxacin kills Chlamydia pneumoniae and modulates interleukin 6 production by HEp-2 cells.

BACKGROUND: Chlamydia pneumoniae is known to cause acute respiratory infection and more recently it has been studied as a pathogen causing inflammatory changes in chronic diseases such as atherosclerosis. This study addresses the antichlamydial effect of levofloxacin and its role in modulation of a proinflammatory cytokine IL-6 production by uninfected and infected HEp-2 cells. METHODS: HEp-2 cell monolayers were infected with previously prepared and frozen aliquots of C.pneumoniae [1 x 10(3) inclusion-forming units (IFU)/ml] by centrifugation for 30 min and incubation at 37 degrees C for 1 h. Infected monolayers were treated with levofloxacin (3 or 8 microg/ml) immediately after infection (0 h) or 24 h after infection. Monolayers were examined daily for 96 h after infection by counting inclusions with fluorescently labeled antichlamydial monoclonal antibody. Aliquots of disrupted monolayers were titrated to determine the numbers of viable C. pneumoniae IFU/ml. IL-6 concentrations in cell supernatants were determined by ELISA assays. RESULTS: Infected HEp-2 cells produced IL-6. Noninfected HEp-2 cells demonstrated modulation of IL-6 production by levofloxacin. No viable C. Pneumoniae were detected in infected HEp-2 cells when the monolayer was treated with levofloxacin immediately after infection (0 h). In contrast, when cells were treated 24 h after infection, a gradual decline in the number of viable C. pneumoniae occurred; by 96 h into the assay >or=98% of C. pneumoniae were killed. IL-6 concentrations were similar in the supernatants of levofloxacin-treated and nontreated HEp-2 cells. CONCLUSIONS: (1). Levofloxacin is effective in eliminating C. pneumoniae from infected HEp-2 cells; (2). although levofloxacin modulates the production of IL-6 in untreated HEp-2 cells, no evidence for such modulation was observed in HEp-2 cells infected with C. pneumoniae. (3). Presence of viable C. pneumoniae may not be necessary for IL-6 production by infected and treated HEp-2 cells.

Molecular subtyping to detect human listeriosis clusters.

We analyzed the diversity (Simpson's Index, D) and distribution of Listeria monocytogenes in human listeriosis cases in New York State (excluding New York City) from November 1996 to June 2000 by using automated ribotyping and pulsed-field gel electrophoresis (PFGE). We applied a scan statistic (p