Cost and mortality prediction using polymerase chain reaction pathogen detection in sepsis: evidence from three observational trials

Delays in adequate antimicrobial treatment contribute to high cost and mortality in sepsis. Polymerase chain reaction (PCR) assays are used alongside conventional cultures to accelerate the identification of microorganisms. We analyze the impact on medical outcomes and healthcare costs if improved adequacy of antimicrobial therapy is achieved by providing immediate coverage after positive PCR reports.

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.

Molecular diagnostics for gonorrhoea: implications for antimicrobial resistance and the threat of untreatable gonorrhoea

This Essay from Nicola Low and colleagues discusses the importance of the nucleic acid amplification tests for rapid detection of N. gonorrhoeae and its resistance determinants, as well as the importance of ensuring their rational use, as priorities for controlling both gonorrhoea and antimicrobial resistance. Please see later in the article for the Editors' Summary.

Antimicrobial susceptibility of gram-positive udder pathogens from bovine mastitis milk in Switzerland

We evaluated the susceptibility of the gram-positive mastitis pathogens S. aureus, Str. uberis, Str. dysgalactiae, E. faecalis and L. garviae to antibiotics that are of epidemiological interest or are critically important for mastitis therapy and human medicine. Penicillin resistance was found to be most frequent in S. aureus, and nearly 5 % of the Str. uberis strains displayed a decreased susceptibility to this antibiotic. Resistance to aminoglycosides and macrolides was also detected in the strains tested. The detection of methicillin-resistant S. aureus (MRSA) and of a ciprofloxacin-resistant Str. dysgalactiae isolate corroborated the emergence of mastitis pathogens resistant to critically important antibiotics and underscores the importance of susceptibility testing prior to antibiotic therapy. The monitoring of antibiotic susceptibility patterns and antibiogram analyses are strongly recommended for targeted antimicrobial treatment and to avoid the unnecessary use of the latest generation of antibiotics.

Detection, treatment, and prevention of carbapenemase-producing Enterobacteriaceae : Recommendations from an International Working Group

The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has increased during the past 10 years. Its detection is frequently difficult, because they do not always show a minimum inhibitory concentration (MIC) value for carbapenems in the resistance range. Both broth microdilution and agar dilution methods are more sensitive than disk diffusion method, Etest and automated systems. Studies on antimicrobial treatment are based on a limited number of patients; therefore, the optimal treatment is not well established. Combination therapy with two active drugs appears to be more effective than monotherapy. Combination of a carbapenem with another active agent — preferentially an aminoglycoside or colistin — could lower mortality provided that the MIC is #4 mg/l and probably #8 mg/l, and is administered in a higher-dose/prolonged-infusion regimen. An aggressive infection control and prevention strategy is recommended, including reinforcement of hand hygiene, using contact precautions and early detection of CPE through use of targeted surveillance.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

A Multiplex Real-Time PCR with High Resolution Melting Analysis for the Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detecting AMR determinants could provide valuable tools for surveillance, epidemiological studies and to inform individual case management. We developed a fast (<1.5 hrs) SYBR-green based real-time PCR method with high resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully-characterized N. gonorrhoeae strains, 19 commensal Neisseria spp., and an additional panel of 193 gonococcal isolates. Results were compared with culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with non-gonococcal Neisseria species and the detection limit was 10(3)-10(4) gDNA copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity 100%, specificity 90%), cefixime (sensitivity 92%, specificity 94%), azithromycin and spectinomycin (both sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations generating resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens but this method can be used to screen collections of gonococcal isolates for AMR more quickly than with current culture-based AMR testing.