Evaluation of a commercial immunochromatographic assay for rapid routine identification of PBP2a-positive Staphylococcus aureus and coagulase-negative staphylococci.

We evaluated the performance of an immunochromatographic assay (PBP2a Culture Colony Test - Alere™), detecting protein-binding penicillin 2a on staphylococci primary isolates in only 6minutes. The assay is highly sensitive for the direct detection of MRSA on various culture media whereas it requires cefoxitin induction for methicillin-resistant coagulase-negative staphylococci.

Reassessment of the Role of Rapid Antigen Detection Tests in Diagnosis of Invasive Group A Streptococcal Infections.

Rapid antigen detection tests (RADTs) for group A streptococci (GAS) are widely used for diagnosing acute pharyngitis, which has led to a considerable reduction in antibiotic prescriptions over the past decade. Beyond this intended use, their reassessment on invasive samples may be relevant in the management of life-threatening GAS infections. To this end, we evaluated the performances of three RADTs, culture, GAS PCR, and 16S rRNA gene PCR assays, and compared them with a composite gold standard (GAS-PCR assay and/or culture) for the diagnosis of severe GAS infection. A total of 192 specimens from deep-tissue (mostly normally sterile) sites enriched for 75 GAS-positive samples were enrolled in the study. The three evaluated RADTs showed sensitivities ranging from 88.0% to 94.7% versus 98.7% for GAS PCR, 84% for 16S rRNA gene PCR, and 77.3% for culture. The sensitivities of the ImmunoCardSTAT! Strep A test (Meridian Bioscience) and the NADAL Strep A strip (Nal Von Minden) were similar to that of GAS PCR (P= 0.25 and 0.03, respectively) and higher than that of culture (P= 0.001 and 0.006, respectively), whereas the SD Bioline Strep A test strip (Standard Diagnostics) showed a performance similar to that of culture (P= 0.02). The three RADTs detected 10 distinctemmtypes, including a predominance ofemm1 (33.3%),emm89 (10.6%), andemm12 (7.6%). No false-positive results were observed, leading to a specificity of 100% for all the evaluated RADTs. The GAS RADTs turned out to be sensitive, specific, and easy-to-use tools that may aid in the management of invasive GAS infections in 24/7 point-of-care laboratories by enabling early diagnosis and focused therapy.

Direct Identification of Staphylococcus aureus and Determination of Methicillin Susceptibility From Positive Blood-Culture Bottles in a Bact/ALERT System Using Binax Now S. aureus and PBP2a Tests.

Staphylococcus aureus bacteremia is associated with high mortality and morbidity, requiring prompt and appropriate antimicrobial treatment. Therefore, it is important to detect methicillin-resistant S. aureus (MRSA) rapidly from blood cultures. Two immunochromatographic tests, BinaxNow S. aureus and BinaxNow PBP2a, were directly applied to 79 Bact/Alert bottles that were positive for Gram positive cocci in cluster aggregations. Sensitivity and specificity for the identification of S. aureus and determination of methicillin resistance were 94% and 87%, and 100% and 100%, respectively, with less than 30 min of performance time. These tests are efficient and rapid; these tests are valuable alternatives to more sophisticated and expensive methods used in the diagnosis of MRSA bacteremia.

A French multicentric study and review of pulmonary Nocardia spp. in cystic fibrosis patients.

Some bacterial species recovered from the airways of cystic fibrosis (CF) patients are indisputably associated with lung infections, whereas the clinical relevance of others, such as Nocardia spp., remains unclear. Sixteen French CF cases of colonization/infection with Nocardia spp. were reviewed in order to evaluate the epidemiology, the clinical impact and the potential treatment of these bacteria, and results were compared to those of the literature. Five Nocardia species were identified, Nocardia cyriacigeorgica being the major species (50 % of cases). At first isolation, Nocardia was the sole pathogen recovered in six patients. Seven patients presented pulmonary exacerbation. For 12 patients, antimicrobial treatment against Nocardia was started immediately, mainly based on cotrimoxazole (6 of the 12 cases). In this study, we highlight the heterogeneity of the clinical management of Nocardia spp. in CF. Guidelines for the clinical management of Nocardia infections in CF patients are proposed.

Exposure of Staphylococcus aureus to subinhibitory concentrations of ß-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus.

Glycopeptides are known to select for heterogeneous vancomycin-intermediate Staphylococcus aureus (h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 days in vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of ≥90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR, yvqF, SA1703, graRS, walKR, and rpoB) were PCR sequenced; no de novo mutations were observed upon beta-lactam exposure. To determine whether trfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, a trfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistance in vitro. This induction required an intact trfA locus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections.

Rapid detection of Staphylococcus aureus and methicillin resistance in bone and joint infection samples: evaluation of the GeneXpert MRSA/SA SSTI assay.

The GeneXpert MRSA/SA SSTI assay was compared to conventional cultures to detect Staphylococcus aureus and methicillin-resistance from 91 bone and joint infection samples. Sensitivity and specificity were 94.4% and 100%. Three false-positive results were observed, in fact providing from patients known to be infected by S. aureus on the basis of other concomitant osteoarticular samples, which suggests that PCR was more sensitive than culture. This diagnosis accuracy may help shorten toxic and non-optimal empirical therapies such as glycopeptides in case of methicillin-susceptible strains.

Septic arthritis caused by noncapsulated Haemophilus influenzae.

Since the introduction of type b Haemophilus influenzae vaccination, noncapsulated H. influenzae has become responsible for most cases of invasive H. influenzae diseases. In our two cases of septic arthritis, we isolated strains with ß-lactamase-positive amoxicillin-clavulanate resistance (BLPACR). Thus, the increasing prevalence of BLPACR should be taken into account when empirical therapy is chosen for septic arthritis.

Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

The rtxA toxin gene of Kingella kingae: a pertinent target for molecular diagnosis of osteoarticular infections.

Kingella kingae is an emerging osteoarticular pathogen in young children. Its isolation by traditional culture methods remains difficult, underscoring the need to implement other diagnostic methods for its detection and identification, such as nucleic acid amplification tests. Although the genome of this bacterium has not yet been sequenced, a toxin named RTX has been identified. The goal of this study was to develop sensitive, specific, and rapid molecular methods based on the rtxA toxin gene sequence to diagnose this infection. Two real-time PCR assays (SYBR green and TaqMan chemistries) targeting this gene are reported. Sensitivity and specificity were first evaluated successfully with 67 strains: 31 Kingella kingae isolates and 36 strains from other bacterial species. Then, 52 clinical specimens positive or negative by culture and/or PCR (16S rRNA and cpn60 genes) were tested with these assays. A nested PCR assay with subsequent sequencing was also developed to confirm the presence of Kingella kingae isolates in these clinical specimens. The results obtained demonstrate that these assays are accurate for the diagnosis of Kingella kingae infection.

Specific real-time polymerase chain reaction places Kingella kingae as the most common cause of osteoarticular infections in young children.

BACKGROUND: The use of universal 16S rDNA polymerase chain reaction (PCR) has recently shown that the place of Kingella kingae in osteoarticular infections (OAI) in young children has been underestimated, but this technique is not the most sensitive or the most rapid method for molecular diagnosis. We developed a specific real-time PCR method to detect K. kingae DNA and applied it to the etiologic diagnosis of OAI. PATIENTS AND METHODS: All children admitted to a pediatric unit for OAI between January 2004 and December 2005 were enrolled in this prospective study. Culture-negative osteoarticular specimens were tested by 16S rDNA PCR and by K. kingae-specific real-time PCR when sufficient sample remained. RESULTS: By culture alone, a pathogen was identified in 45% of the 131 specimens tested (Staphylococcus aureus, n = 25; K. kingae, n = 17; others, n = 18). 16S rDNA PCR and K. kingae-specific PCR were both applied to 61 of the culture-negative samples. The combination of culture and 16S rDNA PCR identified a pathogen in 61% of cases (K. kingae DNA, n = 16; DNA of other microorganisms, n = 5). Specific real-time PCR identified a further 6 cases caused by K. kingae and confirmed all 16 universal PCR-positive cases, bringing the overall documentation rate to 66%. K. kingae was the leading cause of OAI in this pediatric series (n = 39, 45%), followed by S. aureus (n = 25, 29%) CONCLUSION: The K. kingae-specific real-time PCR places K. kingae as the leading cause of OAI in children at our hospital.

Sustained antibacterial effect of a hand rub gel incorporating chlorhexdine-loaded nanocapsules (Nanochlorex).

In the present study, an original chlorhexidine-loaded nanocapsule-based gel (Nanochlorex) was tested as hand rub gel against the resident skin flora in comparison with 2-propanol 60% (v/v) and 62% (v/v) ethanol-based gel (Purell). After 30-s hand rub, the immediate bactericidal effect of Nanochlorex was found comparable to 2-propanol 60% (v/v) (reduction factor, RF: 0.30+/-0.35 versus 0.38+/-0.55, P>0.05) against aerobic bacteria, whereas the post-values of surviving anaerobes were shown significantly lower from Nanochlorex (P<0.001) and insignificant from 2-propanol 60% (v/v) (P>0.05). Sustained antibacterial effect of Nanochlorex was confirmed against the resident and transient hand flora in two sets of experiment. In the first, the results obtained with the glove-juice technique showed that the bactericidal effect induced by Nanochlorex hand rub persisted throughout 3-h period, while Purell failed to reduce significantly the post-values of surviving bacteria. In the second, repeated artificial contaminations with Staphylococcus epidermidis was carried out onto ex vivo human skin pre-treated by either Nanochlorex or Purell for 5min, then maintained in cell diffusion apparatus for 4h. The log(10) reduction of surviving bacteria was significantly higher with Nanochlorex than that determined with Purell after three successive contaminations (from approximately 5.5 to 1.5 log(10) reduction for Nanochlorex between the first and the third contamination; approximately 1log(10) reduction for Purell throughout the experiment), confirming the sustained antibacterial effect of chlorhexidine-loaded nanocapsule-based gel. The immediate and sustained antibacterial effect of Nanochlorex was explained by chlorhexidine carrier system which improved the drug targeting to bacteria and reduced from osmotic gel further bacterial growth on the skin. Nanochlorex) might constitute a promising approach for hygienic hand disinfection in care practice performing multiple procedures.

Clinical and microbiological features of Inquilinus sp. isolates from five patients with cystic fibrosis.

Patients with cystic fibrosis (CF) may be colonized with unusual gram-negative bacilli whose identification is difficult and clinical impact unclear. We describe the clinical and microbiological features of five colonizations with organisms belonging to the recently described genus Inquilinus in CF patients. Isolates were identified from Burkholderia cepacia selective medium by means of 16S rRNA analysis. All of them were resistant to colistin, penicillins, cephalosporins, and monobactams but exhibited a remarkable susceptibility to imipenem. One of the five patients was transiently colonized with a nonmucoid isolate, whereas the four other patients were persistently colonized over the period of follow-up (8 to 21 months) with mucoid isolates. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA was powerful for strain genotyping and demonstrated the clonality of Inquilinus sp. colonization for the two patients tested. Clinical evolution after the onset of Inquilinus was heterogeneous, but for at least one patient the lung function worsened and eradication of Inquilinus sp. was unsuccessful despite several imipenem courses. Finally, Inquilinus spp. may represent a new threat for CF patients due to their mucoid characteristic, their multiresistant pattern to antibiotics, and their ability to persist in the respiratory tract.

Contribution of a broad range polymerase chain reaction to the diagnosis of osteoarticular infections caused by Kingella kingae: description of twenty-four recent pediatric diagnoses.

BACKGROUND: Microbiologic diagnosis of septic arthritis and osteomyelitis in children is hindered by the less than optimal yield of blood and osteoarticular fluid cultures. PATIENTS AND METHODS: All patients admitted to a pediatric unit for osteoarticular infections (OAI) between January 2001 and February 2004 were enrolled in this prospective study. Osteoarticular fluid and biopsy samples that were negative by conventional culture were tested by polymerase chain reaction (PCR) with universal 16S ribosomal DNA primers. RESULTS: We enrolled 171 children. Culture was positive in 64 cases (37.4%), yielding Kingella kingae in 9 cases. The 107 culture-negative specimens were tested by 16S ribosomal DNA PCR. Fifteen samples (14%) were positive, all for Kingella DNA sequences. K. kingae was the second cause of OAI in this population (30.4%), after Staphylococcus aureus (38%). Patients with Kingella infection diagnosed by culture (9 cases) did not differ from those diagnosed by PCR (15 cases) in terms of their clinical characteristics (including prior antibiotic therapy). The characteristics of the 24 children with arthritis (n = 17) or osteomyelitis (n = 7) were similar to those reported elsewhere. Fever (>38 degrees C) and symptom onset shortly before hospitalization (median, 4.5 days) were significantly associated with arthritis. CONCLUSION: Use of molecular diagnostic methods increases the identification of K. kingae in osteoarticular infections.

Specific identification of Staphylococcus aureus by Staphychrom II, a rapid chromogenic staphylocoagulase test.

We compared the performance of Staphychrom II (International Microbio, Signes, France), a rapid (2-h) chromogenic staphylocoagulase test that uses human prothrombin and protease inhibitors, with those of the reference tube coagulase test (TCT) and the latex agglutination test (LAT) Slidex Staph Plus for the rapid identification of S. aureus. Prospective evaluation with 293 fresh clinical isolates yielded sensitivities, specificities, and predictive and negative predictive values of 98.1, 100, 100, and 95.1%, respectively, for the Staphychrom II test; 98.6, 98.7, 99.6, and 96.3%, respectively, for LAT; and 97.6, 98.7, 99.5, and 93.9%, respectively, for TCT. The perfect specificity of the Staphychrom II test was confirmed by testing 193 collection strains selected because of their potential testing pitfalls. The Staphychrom II test was positive for 90% of the 215 S. aureus strains tested after only 1 h of incubation. The Staphychrom II test was as sensitive as the reference TCT and was 100% specific.