Improved sensitivity for molecular detection of bacterial and Candida infections in blood.

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 whole-blood 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.

Enhanced diagnostic yields of bacteremia and candidemia in blood specimens by PCR-electrospray ionization mass spectrometry.

A prospective study was performed to determine the value of direct molecular testing of whole blood for detecting the presence of culturable and unculturable bacteria and yeasts in patients with suspected bloodstream infections. A total of 464 adult and pediatric patients with positive blood cultures matched with 442 patients with negative blood cultures collected during the same period were recruited during a 10-month study. PCR amplification coupled with electrospray ionization mass spectrometry (PCR-ESI-MS) plus blood culture reached an overall agreement of 78.6% in the detection and species-level identification of bacterial and candidal pathogens. Of 33 culture-negative/PCR-ESI-MS-positive specimens, 31 (93.9%) were judged to be truly bacteremic and/or candidemic based on a medical chart review and analytical metrics. Among the 15 culture-positive specimens in which PCR-ESI-MS detected additional bacterial or yeast species, 66.7% and 20.0% of the additional positive specimens by PCR-ESI-MS were judged to be truly or possibly bacteremic and/or candidemic, respectively. Direct analysis of blood samples by PCR-ESI-MS rapidly detects bacterial and yeast pathogens in patients with bloodstream infections. When used in conjunction with blood culture, PCR-ESI-MS enhances the diagnostics of septicemia by shortening test turnaround time and improving yields.

Obstetric complications in patients with hereditary thrombophilia identified using the LCx microparticle enzyme immunoassay: a controlled study of 5,000 patients.

Factor V Leiden (FVL) and prothrombin (PT) G20210A mutations are associated with increased risk of deep venous thrombosis, pulmonary embolism, and obstetric complications. The development of inexpensive and reliable screening methods will assist in defining subpopulations of patients at risk who should undergo testing. We used a method, developed by Abbott Laboratories (Abbott Park, IL), to study 5,000 pregnant women and evaluated the association of obstetric complications with the presence of the FVL and PT G20210A mutations. We found a statistically significant association between FVL and stillbirth. There were also trends toward an association between FVL and placental abruption and between PT G20210A and intrauterine growth retardation. In addition, an association may exist between PT G20210A and preterm delivery for white women. All other parameters studied, including miscarriage and preeclampsia, did not show a statistically significant association with FVL or PT G20210A. These results confirm the association between genetic thrombophilia and selected obstetric complications.