Performance and impact of a multiplex PCR in ICU patients with ventilator-associated pneumonia or ventilated hospital-acquired pneumonia.

BACKGROUND: Early appropriate antibiotic therapy reduces morbidity and mortality of severe pneumonia. However, the emergence of bacterial resistance requires the earliest use of antibiotics with the narrowest possible spectrum. The Unyvero Hospitalized Pneumonia (HPN, Curetis) test is a multiplex PCR (M-PCR) system detecting 21 bacteria and 19 resistance genes on respiratory samples within 5 h. We assessed the performance and the potential impact of the M-PCR on the antibiotic therapy of ICU patients. METHODS: In this prospective study, we performed a M-PCR on bronchoalveolar lavage (BAL) or plugged telescoping catheter (PTC) samples of patients with ventilated HAP or VAP with Gram-negative bacilli or clustered Gram-positive cocci. This study was conducted in 3 ICUs in a French academic hospital: the medical and infectious diseases ICU, the surgical ICU, and the cardio-surgical ICU. A multidisciplinary expert panel simulated the antibiotic changes they would have made if the M-PCR results had been available. RESULTS: We analyzed 95 clinical samples of ventilated HAP or VAP (72 BAL and 23 PTC) from 85 patients (62 males, median age 64 years). The median turnaround time of the M-PCR was 4.6 h (IQR 4.4-5). A total of 90/112 bacteria were detected by the M-PCR system with a global sensitivity of 80% (95% CI, 73-88%) and specificity of 99% (95% CI 99-100). The sensitivity was better for Gram-negative bacteria (90%) than for Gram-positive cocci (62%) (p = 0.005). Moreover, 5/8 extended-spectrum beta-lactamases (CTX-M gene) and 4/4 carbapenemases genes (3 NDM, one oxa-48) were detected. The M-PCR could have led to the earlier initiation of an effective antibiotic in 20/95 patients (21%) and to early de-escalation in 37 patients (39%) but could also have led to one (1%) inadequate antimicrobial therapy. Among 17 empiric antibiotic treatments with carbapenems, 10 could have been de-escalated in the following hours according to the M-PCR results. The M-PCR also led to 2 unexpected diagnosis of severe legionellosis confirmed by culture methods. CONCLUSIONS: Our results suggest that the use of a M-PCR system for respiratory samples of patients with VAP and ventilated HAP could improve empirical antimicrobial therapy and reduce the use of broad-spectrum antibiotics.

PCR methodology and applications for the detection of human fungal pathogens.

INTRODUCTION: Polymerase chain reaction (PCR) has emerged as a promising technology for the rapid and reliable detection and identification of medical mycoses. Recent technological advancements - including microarray, multiplex PCR with magnetic resonance, and beacon probes - have mitigated the technical difficulties of performing nucleic amplification in fungi, thereby improving the sensitivity and specificity of PCR-based assays. In this paper, we examine current applications of PCR in the diagnosis of human fungal infections and look ahead to emerging techniques that may play a larger role in molecular diagnostics in the future. AREAS COVERED: This review includes a brief overview of the advantages and disadvantages of PCR using various clinical specimens, manual versus automated DNA extraction procedures, panfungal versus specific targets, and spectrum of pathogens detected. This is followed by a brief synopsis of species-specific PCR approaches and a more in-depth look at the obstacles to widespread implementation. Expert commentary: The review concludes with a short perspective for the next five years, including the hurdles to standardization and validation, as well as the role of PCR coupled with electrospray-ionization mass spectrometry (PCR/ESI-MS) or nuclear magnetic resonance for the diagnosis of medical mycoses.

Genotyping and interpretation of STR-DNA: Low-template, mixtures and database matches-Twenty years of research and development.

The introduction of Short Tandem Repeat (STR) DNA was a revolution within a revolution that transformed forensic DNA profiling into a tool that could be used, for the first time, to create National DNA databases. This transformation would not have been possible without the concurrent development of fluorescent automated sequencers, combined with the ability to multiplex several loci together. Use of the polymerase chain reaction (PCR) increased the sensitivity of the method to enable the analysis of a handful of cells. The first multiplexes were simple: 'the quad', introduced by the defunct UK Forensic Science Service (FSS) in 1994, rapidly followed by a more discriminating 'six-plex' (Second Generation Multiplex) in 1995 that was used to create the world's first national DNA database. The success of the database rapidly outgrew the functionality of the original system - by the year 2000 a new multiplex of ten-loci was introduced to reduce the chance of adventitious matches. The technology was adopted world-wide, albeit with different loci. The political requirement to introduce pan-European databases encouraged standardisation - the development of European Standard Set (ESS) of markers comprising twelve-loci is the latest iteration. Although development has been impressive, the methods used to interpret evidence have lagged behind. For example, the theory to interpret complex DNA profiles (low-level mixtures), had been developed fifteen years ago, but only in the past year or so, are the concepts starting to be widely adopted. A plethora of different models (some commercial and others non-commercial) have appeared. This has led to a confusing 'debate' about the 'best' to use. The different models available are described along with their advantages and disadvantages. A section discusses the development of national DNA databases, along with details of an associated controversy to estimate the strength of evidence of matches. Current methodology is limited to searches of complete profiles - another example where the interpretation of matches has not kept pace with development of theory. STRs have also transformed the area of Disaster Victim Identification (DVI) which frequently requires kinship analysis. However, genotyping efficiency is complicated by complex, degraded DNA profiles. Finally, there is now a detailed understanding of the causes of stochastic effects that cause DNA profiles to exhibit the phenomena of drop-out and drop-in, along with artefacts such as stutters. The phenomena discussed include: heterozygote balance; stutter; degradation; the effect of decreasing quantities of DNA; the dilution effect.

Comparison of molecular multiplex and singleplex analysis of IgE to grass pollen allergens in untreated german grass pollen–allergic patients

Antecedentes: El ImmunoCAP ISAC 112, es el único sistema comercial con determinación simultánea de múltiples alérgenos comercializado para el diagnóstico alergológico molecular. No existen estudios comparativos de este sistema con el ImmunoCAP para la determinación de IgE frente a un único alérgeno. Objetivos: Realizar un estudio comparativo para la determinación de IgE específica a alérgenos de polen de gramíneas en pacientes alemanes con alergia a estos pólenes, utilizando los sistemas ISAC IgE y el ImmunoCAP IgE. Métodos: Se estudiaron 101 sueros de adultos con alergia a pólenes de gramíneas, determinando la IgE específica a 8 alérgenos de hierba timotea mediante ImmunoCAP y a 112 alérgenos presentes en la plataforma ISAC. Posteriormente se realizó un análisis estadístico comparativo entre los resultados de ambos sistemas. Resultados: La comparación de los valores de IgE específica frente a los alérgenos de pólenes de gramíneas hallados en los sistemas ISAC e ImmunoCAP mostraron los siguientes coeficientes de correlación: 0.88 (rPhl p 1), 0.96 (rPhl p 2), 0.70 (nPhl p 4), 0.94 (rPhl p 5b), 0.92 (rPhl p 6), 0.85 (rPhl p 11) y 0.78 (rPhl p12). Conclusiones: El diagnóstico molecular con el Sistema ISAC guarda buena correlación con los resultados del ImmunoCAP para los alérgenos de hierba timotea presentes en ambas plataformas (AU)

Background: The ImmunoCAP ISAC 112 platform is the only commercially available molecular allergy IgE multiplex test. Data on the comparison of this rather novel test with the molecular singleplex ImmunoCAP IgE platform are lacking. Objective: To compare the multiplex ISAC 112 platform and the singleplex ImmunoCAP platform in regard to IgE to grass pollen allergens in untreated grass pollen–allergic patients in Germany. Methods: Serum samples from 101 adults with grass pollen allergy were analyzed for specific IgE (sIgE) to 8 allergenic molecules from timothy grass pollen and to the 112 allergenic molecules included in the ISAC panel. The results for the multiplex and singleplex tests were subsequently analyzed statistically. Results: Comparison of sIgE to grass pollen allergens detected by ISAC 112 and the singleplex ImmunoCAP assay revealed the following correlation coefficients: 0.88 (rPhl p 1), 0.96 (rPhl p 2), 0.70 (nPhl p 4), 0.94 (rPhl p 5b), 0.92 (rPhl p 6), 0.85 (rPhl p 11), and 0.78 (rPhl p12). Conclusion: Molecular testing with ISAC 112 correlates well with the ImmunoCAP platform for respective molecular timothy grass pollen allergens (AU)