Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample inhibition and suitability for detection of low levels of target DNA in random and multiple samples.

Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae.

Early appropriate antibiotic treatment reduces mortality in severe sepsis, but current methods to identify antibiotic resistance still generally rely on bacterial culture. Modern diagnostics promise rapid gene detection, but the apparent diversity of relevant resistance genes in Enterobacteriaceae is a problem. Local surveys and analysis of publicly available data sets suggested that the resistance gene pool is dominated by a relatively small subset of genes, with a very high positive predictive value for phenotype. In this study, 152 Escherichia coli and 115 Klebsiella pneumoniae consecutive isolates with a cefotaxime, ceftriaxone and/or ceftazidime minimum inhibitory concentration (MIC) of ≥ 2 µg/mL were collected from seven major hospitals in Sydney (Australia) in 2008-2009. Nearly all of those with a MIC in excess of European Committee on Antimicrobial Susceptibility Testing (EUCAST) resistance breakpoints contained one or more representatives of only seven gene types capable of explaining this phenotype, and this included 96% of those with a MIC ≥ 2 µg/mL to any one of these drugs. Similarly, 97% of associated gentamicin-non-susceptibility (MIC ≥ 8 µg/mL) could be explained by three gene types. In a country like Australia, with a background prevalence of resistance to third-generation cephalosporins of 5-10%, this equates to a negative predictive value of >99.5% for non-susceptibility and is therefore suitable for diagnostic application. This is an important proof-of-principle that should be tested in other geographic locations.

Bacteria commonly isolated from keratitis specimens retain antibiotic susceptibility to fluoroquinolones and gentamicin plus cephalothin.

PURPOSE: Patients presenting with presumed infective keratitis were studied to determine predisposing factors, the current susceptibilities of the bacterial isolates to a range of relevant antibiotics, the success rate of topical antibiotic treatment of keratitis and predictors of failure of topical therapy. METHODS: Corneal scrapings taken from patients who presented between January 2002 and December 2003 to the Sydney Eye Hospital Emergency Department with keratitis were cultured. The minimum inhibitory concentration of selected antibiotics was determined for each bacterial isolate using an agar dilution technique. RESULTS: One hundred and twelve consecutive patients presented with corneal ulcers. Forty-seven of the 112 (42%) patients had a growth from the corneal scraping. Potential predisposing factors were identified in 64% of patients, most frequently contact lens wear (36% of patients). Coagulase-negative staphylococci were the most common species isolated. Other common organisms isolated include Pseudomonas aeruginosa, Corynebacterium spp., Staphylococcus aureus and Streptococcus spp. CONCLUSIONS: Most microorganisms isolated from patients with bacterial keratitis showed susceptibility to ciprofloxacin and aminoglycosides. Cephalothin plus aminoglycoside constituted an effective initial broad-spectrum antibiotic combination. The success rate of topical antibiotic treatment of corneal abscess is 89%. Predictors of failure include older age group, medium or large ulcer, culture-negative keratitis, hypopyon and poor visual acuity.