The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood.

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours.The IRIDICA BAC BSI Assay is not available in the United States

Linearity.

1<p>Only linear in samples without environmental air background.</p

Comprehensive Biothreat Cluster Identification by PCR/Electrospray-Ionization Mass Spectrometry

<div><p>Technology for comprehensive identification of biothreats in environmental and clinical specimens is needed to protect citizens in the case of a biological attack. This is a challenge because there are dozens of bacterial and viral species that might be used in a biological attack and many have closely related near-neighbor organisms that are harmless. The biothreat agent, along with its near neighbors, can be thought of as a <em>biothreat cluster</em> or a <em>biocluster</em> for short. The ability to comprehensively detect the important biothreat clusters with resolution sufficient to distinguish the near neighbors with an extremely low false positive rate is required. A technological solution to this problem can be achieved by coupling biothreat group-specific PCR with electrospray ionization mass spectrometry (PCR/ESI-MS). The biothreat assay described here detects ten bacterial and four viral biothreat clusters on the NIAID priority pathogen and HHS/USDA select agent lists. Detection of each of the biothreat clusters was validated by analysis of a broad collection of biothreat organisms and near neighbors prepared by spiking biothreat nucleic acids into nucleic acids extracted from filtered environmental air. Analytical experiments were carried out to determine breadth of coverage, limits of detection, linearity, sensitivity, and specificity. Further, the assay breadth was demonstrated by testing a diverse collection of organisms from each biothreat cluster. The biothreat assay as configured was able to detect all the target organism clusters and did not misidentify any of the near-neighbor organisms as threats. Coupling biothreat cluster-specific PCR to electrospray ionization mass spectrometry simultaneously provides the breadth of coverage, discrimination of near neighbors, and an extremely low false positive rate due to the requirement that an amplicon with a precise base composition of a biothreat agent be detected by mass spectrometry.</p> </div

Organisms tested in analytical studies.

The four core organisms are shown in bold, with LODs in blood and buffer shown in parentheses (blood LOD in CFU per ml / buffer LOD in CFU per ml). The LOD of 19 further organisms in buffer are also shown in parentheses. Remaining organisms shown in blue and purple were tested and detected in 2/2 replicates at either 100 CFU/ml (all except Bacteroides fragilis) or 200 CFU/ml (Bacteroides fragilis). *Indicates bioinformatic “worst-case scenario” organisms, for which the broad-spectrum primers used in the IRIDICA BAC BSI Assay are the least well-matched.</p

Limits of detection and false negative rates.

<p>Limits of detection and false negative rates.</p

Linearity of response of the biothreat assay.

<p><i>B. anthracis</i> DNA was spiked into AE buffer (Clean) or an extract from an environmental air filter (Dirty) at concentrations ranging from 0–1000 GE/well. The reported response from the PLEX-ID system was linear over the entire concentration range tested in both cases.</p

Biothreat cluster detection primer pair and target sites.

<p>Biothreat cluster detection primer pair and target sites.</p

Theoretical and experimental data for <i>B anthracis</i> cluster.¹

1<p>Bolded base counts indicate SNP variants compared to the primary signature at that locus.</p>2<p><i>B. anthracis</i> and near-neighbor organisms were obtained from USAMRIID; details provided in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036528#pone-0036528-t001" target="_blank">Table 1</a>.</p

Detailed results from samples with additional detections.

<p>Detailed data from all samples yielding unmatched positive results by either culture or IRIDICA BAC BSI Assay analysis in the presence of matched or unmatched detections by the other technology (additional detections). The matched positive results shown here in the second column are also represented in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158186#pone.0158186.t003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158186#pone.0158186.t004" target="_blank">4</a>, while the additional detections shown here in the rightmost two columns are not, because they have no valid comparator. See discussion of additional detections in text.</p