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Automated extraction improves multiplex molecular detection of infection in septic patients.

ABSTRACT: Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the automated MagNA Pure compact nucleic acid isolation kit-I (Roche Applied Science, GmbH) as an alternative to conventional SeptiFast extraction. For the purposes of this study, we evaluate extraction in order to demonstrate the feasibility of automation. Finally, a prospective observational study was done using 106 clinical samples obtained from 76 patients in our ICU. Both extraction methods were used in parallel to test the samples. When molecular detection test results using both manual and automated extraction were compared with the data from blood cultures obtained at the same time, the results show that SeptiFast with the alternative MagNA Pure compact extraction not only shortens the complete workflow to 3.57 hrs., but also increases sensitivity of the molecular assay for detecting infection as defined by positive blood culture confirmation.

SUBMITTER: Regueiro BJ

PROVIDER: S-EPMC2954180 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Automated extraction improves multiplex molecular detection of infection in septic patients.

Regueiro Benito J BJ Varela-Ledo Eduardo E Martinez-Lamas Lucia L Rodriguez-Calviño Javier J Aguilera Antonio A Santos Antonio A Gomez-Tato Antonio A Alvarez-Escudero Julian J Alvarez-Escudero Julian J

PloS one 20101013 10

Sepsis is one of the leading causes of morbidity and mortality in hospitalized patients worldwide. Molecular technologies for rapid detection of microorganisms in patients with sepsis have only recently become available. LightCycler SeptiFast test M(grade) (Roche Diagnostics GmbH) is a multiplex PCR analysis able to detect DNA of the 25 most frequent pathogens in bloodstream infections. The time and labor saved while avoiding excessive laboratory manipulation is the rationale for selecting the a ...[more]

PMID: 20967222

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Project description:Variability in expressive and receptive language, difficulty with pragmatic language, and prosodic difficulties are all features of autism spectrum disorder (ASD). Quantifying language and voice characteristics is an important step for measuring outcomes for autistic people, yet clinical measurement is cumbersome and costly. Using natural language processing (NLP) methods and a harmonic model of speech, we analyzed language transcripts and audio recordings to automatically classify individuals as ASD or non-ASD. One-hundred fifty-eight participants (88 ASD, 70 non-ASD) ages 7 to 17 were evaluated with the autism diagnostic observation schedule (ADOS-2), module 3. The ADOS-2 was transcribed following modified SALT guidelines. Seven automated language measures (ALMs) and 10 automated voice measures (AVMs) for each participant were generated from the transcripts and audio of one ADOS-2 task. The measures were analyzed using support vector machine (SVM; a binary classifier) and receiver operating characteristic (ROC). The AVM model resulted in an ROC area under the curve (AUC) of 0.7800, the ALM model an AUC of 0.8748, and the combined model a significantly improved AUC of 0.9205. The ALM model better detected ASD participants who were younger and had lower language skills and shorter activity time. ASD participants detected by the AVM model had better language profiles than those detected by the language model. In combination, automated measurement of language and voice characteristics successfully differentiated children with and without autism. This methodology could help design robust outcome measures for future research. LAY SUMMARY: People with autism often struggle with communication differences which traditional clinical measures and language tests cannot fully capture. Using language transcripts and audio recordings from 158 children ages 7 to 17, we showed that automated, objective language and voice measurements successfully predict the child's diagnosis. This methodology could help design improved outcome measures for research.

Project description:A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

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Automated extraction improves multiplex molecular detection of infection in septic patients.

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Automated extraction improves multiplex molecular detection of infection in septic patients.

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