New technology for rapid molecular diagnosis of bloodstream infections

Figures & data

Figure 1. Identification and genotyping of microbes by PCR/ESI/mass spectrometry.

ESI: Electrospray ionization.

Figure 2. Bacterial phylogenetic tree showing the primer coverage of the PCR/electrospray ionization/mass spectrometry assay.

The primer coverage of the rDNA is represented by the gray background. Exceptions are indicated by red boxes.

Figure 3. (A) Base composition signatures of Candida spp. Columns correspond to the three chromosomal targets and the mitochondrial rDNA target. AGCT counts for each target are shown in the boxes. (B) Color coding. To create this visualization, base compositions from each primer pair (A, G, C, T) were first mapped into a 3D space according to their relative proportions of G+C, A+C and A+G. This enabled the comparison of base compositions representing amplicons of different lengths, while reducing to three the number of independent axes. For each axis, one end is in turn associated with a primary color; thus, pure hues of cyan, magenta and yellow are associated with the maximum values of G+C, A+C and A+G, respectively. Conversely, the complementary colors are found on the other end of these axes, so pure hues of red, green, and blue are associated with the minimum values of G+C, A+C and A+G, respectively (or equivalently, with the maximum values of A+T, G+T and C+T, respectively). In between, the median values are associated with white on each one of the three axes. Individual color components can thus be extrapolated for each cell, blending the amounts of cyan/white/red, magenta/white/green and yellow/white/blue that correspond to the cell’s G+C, A+C and A+G metrics, respectively. Black ink was added for base compositions representing amplicons whose lengths differ from the consensus length at that particular locus. The process is repeated for each one of the nine loci independently and colors cannot be compared from column to column.

Figure 4. Visualization of the diversity of base composition signatures.

Colored cells represent individual base-composition signatures found for the 332 organisms tested (rows) in the nine loci (columns) that were used for broad bacterial identification. Row height occasionally varies as two or three distinct base counts may be found within the same locus for some organisms. Cells are left blank when no signature was found for the corresponding organism and locus.

Figure 5. Spectra of a sample from a patient with a mixed infection.

A blood specimen from a patient co-infected with Staphylococcus aureus containing a mecA gene and Pseudomonas aeruginosa as analyzed by PCR/electrospray ionization/mass spectrometry. Individual spectra from six of the nine bacteria-targeted amplifications are shown. M/Z was converted to the M domain and the scales were selected to visualize all detected peaks.

Figure 6. Frequency of organisms identified by the microbiology laboratory from seven hospitals.

CoNS: Coagulase-negative staphylococci.

Figure 7. PCR/electrospray ionization/mass spectrometry analysis of samples from patients with suspected tick-borne infections.

Pie chart showing the distribution of pathogens detected by PCR/ESI-MS from patients with suspected tick-borne infections. Of the 213 specimens tested, 40 (18.8%) tested positive for Ehrlichia chaffeensis or Ehrlichia ewingii.

Table 1. Needs and current status of methods to identify bloodstream infections.

Need	Current status			Future opportunity
	Culture	Molecular methods		Molecular methods
Identify all bacterial and fungal pathogens	Identifies only culturable organisms	Varies with method from 25 frequently cultured organisms to panbacterial; limited or no fungal [4,36,37]		Panbacterial and panfungal identification
High sensitivity: bloodstream infections can be caused by less than 10 CFU/ml in adults [8–10]	Blood cultures are negative in >50% of clinically sepsis cases [3]	Mixed results. Blood culture is more sensitive in some cases, but molecular methods identify organisms missed by culture; reported sensitivities as low as 3 CFU/ml [36,37]		Sensitivity similar to culture but with panbacterial and panfungal identification
Rapid identification; mortality increases hourly in the absence of appropriate antimicrobial therapy [2]	Requires 1–5 days	Requires 1 day [40]		<1 h
Antimicrobial resistance determination	Requires 1 additional day after obtaining culture isolate	A few major resistance determinants measured [12,19,20,22,25,30]		Significant opportunity with future understanding of molecular mechanisms
Quantitative assessment of pathogen load; load correlates with disease severity [7,11]	Quantitative culture methods too difficult for routine practice	Not quantitative		Quantitative results
Low labor requirements, automated	Labor intensive	Labor intensive		Fully automated

CFU: Colony-forming unit.

Table 2. Current molecular tests for bloodstream infections.

Company	Product	Status	Sample volume	Specimen preparation	Enrichment	Amplification	Detection	Bacteria	Fungi	Ref.
Roche	SeptiFast^®	CE marked	1.5 ml	Manual	None	Broad-range PCR	Fluorescent probes	20 species	Candida and Aspergillus spp.	[39–47]
SIRS-Lab	LOOXSTER^®/VYOO™	CE marked	5 ml	Manual	Microbial DNA enrichment via affinity	Broad-range PCR	Gels	40 species, 5 antibiotic determinants	Candida spp., Aspergillus and panfungal	[54]
MolZym	SeptiTest	CE marked	5 ml	Manual	Microbial DNA enrichment via selective lysis	Broad-range PCR	Gels, then sequencing	>300 species	Candida spp. and Cryptococcus	[53–56]
Abbott/Ibis	PLEX-ID BAC Spectrum	In clinical studies	1.5 ml	Magnetic beads on KingFisher	None	Broad-range PCR	Mass spectrometry	>300 species, 3 antibiotic determinants	Candida speciation	[58,59,61,68]

Table 3. Comparison of PCR/electrospray ionization/mass spectrometry results for patients infected with non-Ehrlichia spp. with clinical diagnosis and the results of the laboratory confirmations.

Gender	Age (years)	PCR/ESI-MS result	Microbial genomes detected per well	Clinical diagnosis	Laboratory confirmation
Female	11	Rickettsia rickettsii	30	Likely tick-borne illness with dehydration and myalgia	Serum collected at acute phase weak positive for rickettsial IgM but negative for IgG
Male	49	Rickettsia rickettsii	>1000	Clinical findings consistent with Rocky Mountain Spotted Fever	Serum rickettsial IgG titer of 1:512
Female	9 months	Rickettsia rickettsii	360,110	Sepsis with multiorgan failure secondary to Rocky Mountain Spotted Fever	Acuter serum was negative, but convalescent was positive for rickettsial IgG and IgM
Male	71	Pseudomonas aeruginosa	>250	Bacteremia, aspergilosis	Pseudomonas aeruginosa recovered from blood culture
Female	19	Neisseria meningitidis	>250	Bacterial meningitis, sepsis	Neisseria meningitidis recovered from blood culture
Male	47	Bacteroides spp.	>140	Diverticulitis, retroperitoneal abscess	Blood culture was negative
Male	2	Staphylococcus aureus	60	Sepsis, septic arthritits, osteromyelitis	MRSA recovered from blood culture

ESI-MS: Electrospray ionization/mass spectrometry; MRSA: Methicillin-resistant Staphylococcus aureus.

Adapted with permission from [68].

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