Broad-Range Bacterial Detection and the Analysis of Unexplained Death and Critical Illness

Nikkari, Simo; Lopez, Fred A.; Lepp, Paul W.; Cieslak, Paul R.; Ladd-Wilson, Stephen; Passaro, Douglas J.; Danila, Richard; Relman, David A.

doi:10.3201/eid0802.010150

Cited by 85 publications

(54 citation statements)

References 30 publications

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“…Recently, PCR-based assays have become available to provide an early and accurate diagnosis of bacterial meningitis (2,10,12,14,22,30,33,35,39,42,45,46,51,54,60).…”

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confidence: 99%

Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

Schuurman¹,

Boer²,

et al. 2004

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We have evaluated the use of a broad-range PCR aimed at the 16S rRNA gene in detecting bacterial meningitis in a clinical setting. To achieve a uniform DNA extraction procedure for both gram-positive and gram-negative organisms, a combination of physical disruption (bead beating) and a silica-guanidiniumthiocyanate procedure was used for nucleic acid preparation. To diminish the risk of contamination as much as possible, we chose to amplify almost the entire 16S rRNA gene. The analytical sensitivity of the assay was approximately 1 ؋ 10 2 to 2 ؋ 10 2 CFU/ml of cerebrospinal fluid (CSF) for both gram-negative and grampositive bacteria. In a prospective study of 227 CSF samples, broad-range PCR proved to be superior to conventional methods in detecting bacterial meningitis when antimicrobial therapy had already started. Overall, our assay showed a sensitivity of 86%, a specificity of 97%, a positive predictive value of 80%, and a negative predictive value of 98% compared to culture. We are currently adapting the standard procedures in our laboratory for detecting bacterial meningitis; broad-range 16S ribosomal DNA PCR detection is indicated when antimicrobial therapy has already started at time of lumbar puncture or when cultures remain negative, although the suspicion of bacterial meningitis remains.

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“…Recently, PCR-based assays have become available to provide an early and accurate diagnosis of bacterial meningitis (2,10,12,14,22,30,33,35,39,42,45,46,51,54,60).…”

mentioning

confidence: 99%

Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

Schuurman¹,

Boer²,

et al. 2004

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“…Another drawback is that PCR products from samples of patients with multibacterial infections, or from sites containing multiple bacterial species, cannot be directly analyzed by DNA sequencing. Extensive experiments, including molecular cloning followed by sequence analysis of multiple transformants, have been necessary to study polymicrobial infections and clinical specimens containing multiple bacterial species (16,19,29).…”

Section: Discussionmentioning

confidence: 99%

“…However, the kits are not widely used for microbial identification in diagnostic microbiological laboratories due to the time and skill needed to perform the assays, as well as the necessity for costly equipment, e.g., DNA sequencers. The more challenging task of utilizing broad-range PCR analysis directly on clinical specimens has been performed mainly by academic institutions and research laboratories (10,15,19,24,25,35). In the direct approach, the molecular finding is not dependent on viable microorganisms or on those species that cannot readily be cultured by classical methodologies.…”

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confidence: 99%

Use of an Oligonucleotide Array for Laboratory Diagnosis of Bacteria Responsible for Acute Upper Respiratory Infections

Roth¹,

Jalava

Ruuskanen

et al. 2004

J Clin Microbiol

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We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.Modern high-density arrays of DNA probes (microarrays) have proved to be powerful tools to study complex genetic profiles of cellular life. However, only a few experimental assays based on this valuable tool have been introduced for microbiological diagnostics. We present here novel data demonstrating the application of microarray technology combined with broad-range PCR amplification of topoisomerase genes to the diagnosis of bacterial respiratory infections.Broad-range bacterial PCR is based on the use of primers that recognize conserved sequences of bacterial genes encoding essential molecules. The resulting DNA amplicons contain variable regions that provide a basis for the analysis of phylogenetic relationships and identification of different bacterial species. Furthermore, some genetically hypervariable regions provide targets for bacterial-species-specific oligonucleotide sequences. These oligonucleotides that target hypervariable regions may be used in a diagnostic array of probes for detection of bacterial pathogens.The use of broad-range ribosomal DNA (rDNA) PCR technologies for identification of bacterial and fungal culture isolates in the clinical laboratory is well established (3, 28), and there are commercial kits available for that purpose. However, the kits are not widely...

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“…The sequence of the 16S rRNA gene has been used to diagnose and identify bacterial infection in clinical practice (6,27). Some PCR-based assays can be used to identify specific bacterial pathogens (16,32), while broadrange bacterial PCR can detect almost any bacterial species (25,33). The use of broad-range bacterial PCR has a great advantage: it can detect microorganisms that are found less frequently or even unknown causative agents of bacterial origin.…”

mentioning

confidence: 99%

Gram Stain-Specific-Probe-Based Real-Time PCR for Diagnosis and Discrimination of Bacterial Neonatal Sepsis

Chen

et al. 2008

J Clin Microbiol

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Sepsis is a serious disease with high mortality in newborns. It is very important to have a convenient and accurate method for pathogenic diagnosis of neonatal sepsis. We developed a method of simultaneous detection and Gram classification of clinically relevant bacterial pathogens causing sepsis directly from blood samples with Gram stain-specific-probe-based real-time PCR (GSPBRT-PCR). With GSPBRT-PCR, 53 clinically important strains representing 25 gram-positive and 28 gram-negative bacterial species were identified correctly with the corresponding Gram probe. The limits of the GSPBRT-PCR assay in serial dilutions of the bacteria revealed that Staphylococcus aureus could be detected at concentrations of 3 CFU per PCR and Escherichia coli at concentrations as low as 1 CFU per PCR. The GSPBRT-PCR assay was further evaluated on 600 blood specimens from patients with suspicioon of neonatal sepsis and compared to the results obtained from blood cultures. The positive rate of the GSPBRT-PCR array was 50/600 (8.33%), significantly higher than that of blood culture (34/600; 5.67%) (P ‫؍‬ 0.00003). When blood culture was used as a control, the sensitivity of GSPBRT-PCR was 100%, the specificity was 97.17%, and the index of accurate diagnosis was 0.972. This study suggests that GSPBRT-PCR is very useful for the rapid and accurate diagnosis of bacterial infection and that it can have an important impact on the current inappropriate and unnecessary use of antibiotics in the treatment of newborns.

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Broad-Range Bacterial Detection and the Analysis of Unexplained Death and Critical Illness

Cited by 85 publications

References 30 publications

Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

Prospective Study of Use of PCR Amplification and Sequencing of 16S Ribosomal DNA from Cerebrospinal Fluid for Diagnosis of Bacterial Meningitis in a Clinical Setting

Use of an Oligonucleotide Array for Laboratory Diagnosis of Bacteria Responsible for Acute Upper Respiratory Infections

Gram Stain-Specific-Probe-Based Real-Time PCR for Diagnosis and Discrimination of Bacterial Neonatal Sepsis

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