Broad-Spectrum Biosensor Capable of Detecting and Identifying Diverse Bacterial and Candida Species in Blood

Metzgar, David; Frinder, Mark; Lovari, Robert; Toleno, Donna M.; Massire, Christian; Blyn, Lawrence B.; Ranken, Raymond; Carolan, Heather E.; Hall, Thomas A.; Moore, D. J.; Hansen, Christian J.; Sampath, Rangarajan; Ecker, David J.

doi:10.1128/jcm.00966-13

Cited by 37 publications

(35 citation statements)

References 24 publications

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“…The extracted DNA was amplified on the bacterial artificial chromosome (BAC) spectrum assay according to the manufacturer’s instructions. PCR/ESI-MS can identify 3400 bacteria and 40 Candida spp., which are represented in the platform’s signature database [69–71]. A total of 200 μL of extract was used per sample.…”

Section: Methodsmentioning

confidence: 99%

Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques

Romero¹,

Miranda²,

Kusanovic³

et al. 2015

Journal of Perinatal Medicine

209

216

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Introduction The objectives of this study were: 1) to determine the amniotic fluid (AF) microbiology of patients with the diagnosis of clinical chorioamnionitis at term using both cultivation and molecular techniques; and 2) to examine the relationship between intra-amniotic inflammation with and without microorganisms and placental lesions consistent with acute AF infection. Methods The AF samples obtained by transabdominal amniocentesis from 46 women with clinical signs of chorioamnionitis at term were analyzed using cultivation techniques (for aerobic and anerobic bacteria as well as genital mycoplasmas) and broad-range polymerase chain reaction (PCR) coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). The frequency of microbial invasion of the amniotic cavity (MIAC), intra-amniotic inflammation [defined as an AF interleukin 6 (IL-6) concentration ≥ 2.6ng/mL], and placental lesions consistent with acute AF infection (acute histologic chorioamnionitis and/or acute funisitis) were examined according to the results of AF cultivation and PCR/ESI-MS as well as AF IL-6 concentrations. Results 1) Culture identified bacteria in AF from 46% (21/46) of the participants, whereas PCR/ESI-MS was positive formicroorganisms in 59% (27/46) – combining these two tests, microorganisms were detected in 61% (28/46) of patients with clinical chorioamnionitis at term. Eight patients had discordant test results; one had a positive culture and negative PCR/ESI-MS result, whereas seven patients had positive PCR/ESI-MS results and negative cultures. 2) Ureaplasma urealyticum (n = 8) and Gardnerella vaginalis (n = 10) were the microorganisms most frequently identified by cultivation and PCR/ESI-MS, respectively. 3) When combining the results of AF culture, PCR/ESI-MS and AF IL-6 concentrations, 15% (7/46) of patients did not have intra-amniotic inflammation or infection, 6.5% (3/46) had only MIAC, 54% (25/46) had microbial-associated intra-amniotic inflammation, and 24% (11/46) had intra-amniotic inflammation without detectable microorganisms. 4) Placental lesions consistent with acute AF infection were significantly more frequent in patients with microbial-associated intra-amniotic inflammation than in those without intra-amniotic inflammation [70.8% (17/24) vs. 28.6% (2/7); P = 0.04]. Conclusion Microorganisms in the AF were identified in 61% of patients with clinical chorioamnionitis at term; 54% had microbial-associated intra-amniotic inflammation, whereas 24% had intra-amniotic inflammation without detectable microorganisms.

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Section: Methodsmentioning

confidence: 99%

Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques

Romero¹,

Miranda²,

Kusanovic³

et al. 2015

Journal of Perinatal Medicine

209

216

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“…The specimens were kept at 4°C refrigeration within 30 min of collection, shipped at 4°C, and kept frozen until analysis. The blood samples were lysed in the presence of 665 l lysis buffer (Abbott Molecular, Des Plaines, IL) 100 mM Tris solution containing guanidinium thiocyanate and detergent, 145 l of 10% bovine serum albumin (BSA) containing a pumpkin DNA extraction control (24), and 3 g of 0.2-mm yttria-stabilized zirconium oxide beads using a large-volume bead mill homogenizer (under development at Ibis Biosciences, Abbott, Carlsbad, CA, and Omni International, Kennesaw, GA) (speed, 6.6 m/s; three 90-s cycles with 20-s dwell time). The sample tubes were centrifuged at 3,220 ϫ g for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…associated with human infections, including unculturable organisms, directly from blood specimens (21)(22)(23)(24)(25)(26)(27). The assay can also detect three classes of antibiotic resistance markers associated with resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (bla KPC ).…”

mentioning

confidence: 99%

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

et al. 2014

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The rapid identification of bacteria and fungi directly from the blood of patients with suspected bloodstream infections aids in diagnosis and guides treatment decisions. The development of an automated, rapid, and sensitive molecular technology capable of detecting the diverse agents of such infections at low titers has been challenging, due in part to the high background of genomic DNA in blood. PCR followed by electrospray ionization mass spectrometry (PCR/ESI-MS) allows for the rapid and accurate identification of microorganisms but with a sensitivity of about 50% compared to that of culture when using 1-ml wholeblood specimens. Here, we describe a new integrated specimen preparation technology that substantially improves the sensitivity of PCR/ESI-MS analysis. An efficient lysis method and automated DNA purification system were designed for processing 5 ml of whole blood. In addition, PCR amplification formulations were optimized to tolerate high levels of human DNA. An analysis of 331 specimens collected from patients with suspected bloodstream infections resulted in 35 PCR/ESI-MS-positive specimens (10.6%) compared to 18 positive by culture (5.4%). PCR/ESI-MS was 83% sensitive and 94% specific compared to culture. Replicate PCR/ESI-MS testing from a second aliquot of the PCR/ESI-MS-positive/culture-negative specimens corroborated the initial findings in most cases, resulting in increased sensitivity (91%) and specificity (99%) when confirmed detections were considered true positives. The integrated solution described here has the potential to provide rapid detection and identification of organisms responsible for bloodstream infections.

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“…The extracted DNA was amplified by the previously described broad bacteria and candida detection assay, according to the manufacturer's instructions. PCR/ESI/MS can identify 3,400 bacteria and 40 Candida spp., which are represented in the platform's signature database (40).…”

Section: Detection Of Microorganisms With Molecular Methodsmentioning

confidence: 99%

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome

Maddipati

Romero

Chaiworapongsa

et al. 2016

Journal of Lipid Research

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Broad-Spectrum Biosensor Capable of Detecting and Identifying Diverse Bacterial and Candida Species in Blood

Cited by 37 publications

References 24 publications

Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques

Clinical chorioamnionitis at term I: microbiology of the amniotic cavity using cultivation and molecular techniques

Improved Sensitivity for Molecular Detection of Bacterial and Candida Infections in Blood

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome

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