Highly Sensitive Detection of Staphylococcus aureus Directly from Patient Blood

Background: Rapid detection of bloodstream infections (BSIs) can be lifesaving. We investigated the sample processing and assay parameters necessary for highly-sensitive detection of bloodstream bacteria, using Staphylococcus aureus as a model pathogen and an automated fluidic sample processing – polymerase chain reaction (PCR) platform as a model diagnostic system. Methodology/Principal Findings: We compared a short 128 bp amplicon hemi-nested PCR and a relatively shorter 79 bp amplicon nested PCR targeting the S. aureus nuc and sodA genes, respectively. The sodA nested assay showed an enhanced limit of detection (LOD) of 5 genomic copies per reaction or 10 colony forming units (CFU) per ml blood over 50 copies per reaction or 50 CFU/ml for the nuc assay. To establish optimal extraction protocols, we investigated the relative abundance of the bacteria in different components of the blood (white blood cells (WBCs), plasma or whole blood), using the above assays. The blood samples were obtained from the patients who were culture positive for S. aureus. Whole blood resulted in maximum PCR positives with sodA assay (90 % positive) as opposed to cell-associated bacteria (in WBCs) (71 % samples positive) or free bacterial DNA in plasma (62.5 % samples positive). Both the assays were further tested for direct detection of S. aureus in patient whole blood samples that were contemporaneous culture positive. S. aureus was detected in 40/45 of culture-positive patients (sensitivity 89%, 95 % CI 0.75–0.96) and 0/59 negative controls with the sodA assay (specificit

Analytical sensitivity of the <i>nuc</i> and <i>sodA</i> assays for the detection of <i>Staphylococcus aureus</i> genomic DNA (A) and <i>S. aureus</i> cells spiked in blood (B).

<p>Analytical sensitivity of the <i>nuc</i> and <i>sodA</i> assays for the detection of <i>Staphylococcus aureus</i> genomic DNA (A) and <i>S. aureus</i> cells spiked in blood (B).</p

Blood components study processing schematic.

<p>CBC blood from patients with blood culture positive for <i>S. aureus</i> was divided into 1 ml each for detection of <i>S. aureus</i> load in plasma, white blood cells (WBCs) and whole blood. Plasma was processed using a column based resin (CBR) cartridge. WBCs and whole blood was processed in a filter based (FB) cartridge.</p

Flow diagram of the fluidic steps involved in sample processing and PCR amplification for target genes.

<p>Flow diagram of the fluidic steps involved in sample processing and PCR amplification for target genes.</p

Detection of bacterial load in different blood components by <i>nuc</i> (A) and <i>sodA</i> (B) assays.

<p>Each Ct data point (open circles) indicates individual patient samples tested in each blood component (one color for each patient sample). The horizontal line indicates median Ct values. Solid color bars in green (<i>nuc</i> assay) and orange (<i>sodA</i> assay) represent the corresponding % positive on the secondary axis. An “n”, indicates the number of samples analyzed. WBC, indicates white blood cells.</p

Sensitivity and specificity of <i>nuc</i> and <i>sodA</i> assays compared to blood culture as tested with patient blood samples.

<p>Sensitivity and specificity of <i>nuc</i> and <i>sodA</i> assays compared to blood culture as tested with patient blood samples.</p

Disappearance of Vaccine-Type Invasive Pneumococcal Disease and Emergence of Serotype 19A in a Minority Population with a High Prevalence of Human Immunodeficiency Virus and Low Childhood Immunization Rates▿

We analyzed the epidemiology of invasive pneumococcal disease (IPD) following introduction of pneumococcal conjugated vaccine in an urban population with a 2% human immunodeficiency virus (HIV) prevalence and history of low childhood immunization rates. We observed near-elimination of vaccine-type IPD. Substantial disease remains due to non-vaccine-type pneumococci, highlighting the need to increase pneumococcal immunization among HIV-infected adults

Rapid Detection of Mycobacterium tuberculosis and Rifampin Resistance by Use of On-Demand, Near-Patient Technology▿ † ‡

Current nucleic acid amplification methods to detect Mycobacterium tuberculosis are complex, labor-intensive, and technically challenging. We developed and performed the first analysis of the Cepheid Gene Xpert System's MTB/RIF assay, an integrated hands-free sputum-processing and real-time PCR system with rapid on-demand, near-patient technology, to simultaneously detect M. tuberculosis and rifampin resistance. Analytic tests of M. tuberculosis DNA demonstrated a limit of detection (LOD) of 4.5 genomes per reaction. Studies using sputum spiked with known numbers of M. tuberculosis CFU predicted a clinical LOD of 131 CFU/ml. Killing studies showed that the assay's buffer decreased M. tuberculosis viability by at least 8 logs, substantially reducing biohazards. Tests of 23 different commonly occurring rifampin resistance mutations demonstrated that all 23 (100%) would be identified as rifampin resistant. An analysis of 20 nontuberculosis mycobacteria species confirmed high assay specificity. A small clinical validation study of 107 clinical sputum samples from suspected tuberculosis cases in Vietnam detected 29/29 (100%) smear-positive culture-positive cases and 33/39 (84.6%) or 38/53 (71.7%) smear-negative culture-positive cases, as determined by growth on solid medium or on both solid and liquid media, respectively. M. tuberculosis was not detected in 25/25 (100%) of the culture-negative samples. A study of 64 smear-positive culture-positive sputa from retreatment tuberculosis cases in Uganda detected 63/64 (98.4%) culture-positive cases and 9/9 (100%) cases of rifampin resistance. Rifampin resistance was excluded in 54/55 (98.2%) susceptible cases. Specificity rose to 100% after correcting for a conventional susceptibility test error. In conclusion, this highly sensitive and simple-to-use system can detect M. tuberculosis directly from sputum in less than 2 h