New technology for rapid molecular diagnosis of

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New technology for rapid molecular diagnosis of bloodstream infections Expert Rev. Mol. Diagn. 10(4), 399–415 (2010)

David J Ecker1, Rangarajan Sampath1, Haijing Li2, Christian Massire1, Heather E Matthews1, Donna Toleno1, Thomas A Hall1, Lawrence B Blyn1, Mark W Eshoo1, Raymond Ranken1, Steven A Hofstadler1 and Yi-Wei Tang†2

Technologies for the correct and timely diagnosis of bloodstream infections are urgently needed. Molecular diagnostic methods have yet to have a major impact on the diagnosis of bloodstream infections; however, new methods are being developed that are beginning to address key issues. In this article, we discuss the key needs and objectives of molecular diagnostics for bloodstream infections and review some of the currently available methods and how these techniques meet key needs. We then focus on a new method that combines nucleic acid amplification with mass spectrometry in a novel approach to molecular diagnosis of bloodstream infections.

Ibis Biosciences, a subsidiary of Abbott Molecular, Inc., 2251 Faraday Ave, Carlsbad, CA 92008, USA 2 Molecular Infectious Diseases Laboratory, Vanderbilt University Hospital, 4605 TVC, 1161 21st Ave South, Nashville, TN 37232-5310, USA † Author for correspondence: Tel.: +1 615 322 0126 Fax: +1 615 343 8420 [email protected]

Need for improving current diagnostic tools for bloodstream infections

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10.1586/ERM.10.24

1

Keywords : antibiotic resistance • bacteria • candidemia • Ibis • molecular diagnosis • PCR/electrospray ionization mass spectrometry • PLEX-ID • sepsis • systemic inflammatory response syndrome

There is a major unmet need to shorten and improve current laboratory procedures for the detection and identification of microorganisms responsible for bloodstream infections. An ideal diagnostic technology would identify the infecting organism(s) and also the determinants of antibiotic resistance in a timely manner so that appropriate therapy could begin as soon as possible [1] . In the case of the most serious bloodstream infections associated with septic shock, speed is of the essence. It has been shown that the risk of mortality in septic shock increases substantially by the hour if appropriate antimicrobial therapy is delayed. For patients with septic shock, Kumar et  al. have reported that each hour of delay in effective therapy is associated with a 7.6% decrease in survival [2] . The survival rate of severe sepsis goes from approximately 80% if effective therapy is given within the first hour of the onset of symptoms to less than 10% if effective therapy is not given by 24 h. The International Committee on Surviving Sepsis has proposed a ‘strong recommendation’ that patients who show symptoms of sepsis be treated empirically within the first hour with broad-spectrum antibiotics, even though a more targeted therapy would be preferred [3] . The committee also

strongly recommended that blood be drawn before administration of anti­microbial therapy so that therapy can be appropriately targeted when culture results are available. The current gold standard of bloodstream microbial detection and identification is automatic, continuous monitoring liquid culture, followed by Gram stain, subculturing and use of phenotypic methods to identify the organism and its susceptibilities. A major limitation to culture is the time required to complete the process, which ranges from 1 to 5 days or more. This timeline is inconsistent with the need to obtain rapid answers to guide therapy. In addition, culture methods miss fastidious organisms that are difficult or impossible to culture. Culture can also be confounded if antibiotics are administered before the blood is sampled. Blood cultures are reported to be negative in more than 50% of the cases where true bacterial or fungal sepsis is believed to exist [3,4] . This has driven the recommendation by the International Sepsis Committee that decisions on antibiotic administration, changes in therapy or its discontinuation be based on clinical judgment rather than culture results. Thus, blood culture is not an ideal gold standard. The results come too late, are incomplete and are potentially misleading, to the point where the recommendations are to ignore them in many cases.

© Ibis Biosciences, Inc.

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Ecker, Sampath, Li et al.

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Potential for molecular methods

It is remarkable that in this modern era of molecular technology, no molecular method has replaced or is even used broadly as an add-on to blood culture in clinical laboratories for the diagnosis of disease caused by infectious organisms [5,6] . Molecular methods have the potential to overcome many limitations of culture (Table 1) . The ideal molecular method would analyze a patient’s blood and provide all the information needed to immediately direct the optimal antimicrobial therapy for bacterial or fungal infections, and could subsequently provide data to assess the therapy by measuring the clearance of microbial nucleic acids from the blood over time. Although no currently available molecular method is sufficiently rapid, accurate or informative to achieve these goals, these should be the long-term objectives for diagnosis of bloodstream infections. An achievable goal in the near term is to analyze blood in parallel with culture methods and identify the pathogens, including unculturable organisms, responsible for infection and some of the key determinants of drug resistance well before the culture results are available. All of the molecular determinants of drug resistance are not yet known, yet genes have been identified in some species, including genes encoding methicillin resistance, vancomycin resistance and carbapenem resistance, that can be measured using molecular methods. If available, quantitative measurements would be broadly used in microbiology. Many years of research studies using quantitative microbiology on solid media have demonstrated that quantitative measurements provide clinically valuable information [7] . For example, bacterial load is predictive of the occurrence of

complications and death [7] . However, the inability to measure bacterial load routinely has precluded a complete understanding of the value of this metric. Quantification of bacteria in blood is difficult to achieve by culture methods and is rarely practiced in clinical laboratories because it requires subsequent plating on solid media rather than incubation of blood in liquid culture. The time required for liquid culture bottles to become positive provides some suggestion of bacterial load, but is a weak quantitative metric and varies with the microbe or microbes present. Molecular tests have the potential to be quantitative. Quantitative molecular measurements have been standard in managing chronic viral infections, such as HIV and hepatitis C (HCV), for many years. Several factors preclude quantitative measurements in bloodstream infections. First, the range of bacterial concentrations in infected patients is broad and the lowend challenges the lower limit of detection of current molecular methods. Many published studies have shown that the number of recoverable colony-forming units (CFU) of bacteria in the blood of adult patients with clinically significant bacteremia is low, typically in the range of 1–30 CFU/ml [8–10] ; in children, the levels of bacteria are substantially higher, in excess of 100 CFU/ml [7] . However, the CFU measured by culture microbiology represents only the viable organisms that survive the plating process and does not count dead cells, cells that cannot form colonies or free microbial DNA that may have been liberated from lysed cells in the blood compartment. Second, sample preparation methods to extract pathogen DNA from whole blood in sufficient purity and quantity for molecular methods are inadequate. Third, infections can by caused by many dozens of different

Table 1. Needs and current status of methods to identify bloodstream infections. Need Identify all bacterial and fungal pathogens

Current status

Future opportunity

Culture

Molecular methods

Molecular methods

Identifies only culturable organisms

Varies with method from 25 frequently cultured organisms to panbacterial; limited or no fungal

Panbacterial and panfungal identification

[4,36,37]

High sensitivity: bloodstream infections can be caused by less than 10 CFU/ml in adults [8–10]

Blood cultures are Mixed results. Blood culture is more sensitive in negative in >50% of some cases, but molecular methods identify clinically sepsis cases [3] 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]

300 species, 3 antibiotic determinants Mass spectrometry Broad-range PCR None Magnetic beads on KingFisher 1.5 ml PLEX-ID BAC Spectrum Abbott/Ibis

In clinical studies

[53–56]

Candida spp. and Cryptococcus >300 species Gels, then sequencing Broad-range PCR Microbial DNA enrichment via selective lysis Manual SeptiTest MolZym

CE marked 5 ml

[54]

Candida spp., Aspergillus and panfungal LOOXSTER® / VYOO™ SIRS-Lab

CE marked 5 ml

Manual

Microbial DNA enrichment via affinity

Broad-range PCR

Gels

40 species, 5 antibiotic determinants

[39–47]

Candida and Aspergillus spp. 20 species Fluorescent probes Broad-range PCR None Manual SeptiFast® Roche

CE marked 1.5 ml

Bacteria Detection Product

Status

Sample volume

Specimen preparation

Enrichment

Amplification 402

Ecker, Sampath, Li et al.

Company

Table 2. Current molecular tests for bloodstream infections.

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Fungi

Ref.

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SeptiFast®

The Roche product SeptiFast® [38] has been available longer than other molecular-based tests and has been evaluated in a number of published clinical studies [39–47] . SeptiFast uses real-time PCR in a nonquantitative mode to identify ten bacteria at the species level, several more at the genus level, as well as five Candida species and Aspergillus fumagatus. This assay reportedly identifies the 25 organisms that account for more than 90% of the culturable pathogens associated with sepsis. No unculturable organisms are identified nor are most of the highly fastidious organisms that are difficult to culture. The SeptiFast procedure involves extraction of nucleic acid from 1.5 ml of whole blood using mechanical lysis with ceramic beads and manual spin column-based nucleic acid purification under a contamination-controlled workflow [38] . Human DNA from white cells in blood and pathogen DNA are isolated together. Nucleic acid preparation is followed by placing 50‑µl aliquots of the elute into three real-time PCR reactions that amplify Gram-positive and Gram-negative bacteria and fungi. Fluorescent probes are used to identify the pathogens. Clinical studies using SeptiFast on blood samples from hospital patient populations with suspected sepsis [39–41,43,46,47] , emergency department patients with suspected sepsis [44] and febrile neutro­penic patients [45,48] have been reported. From these studies, some important themes have emerged. First, using blood culture as a reference ‘gold standard’ to compare molecular methods is problematic. As described earlier, culture fails to identify more than 50% of the cases of sepsis believed to be caused by bacteria or fungi based on clinical and other criteria [3] . Unsurprisingly, SeptiFast consistently identified more positive specimens than blood culture methods. These potential ‘false positives’ were frequently deemed clinically significant based on chart review of clinical data, other analytical evidence of infection or disease severity [39] , and were often subsequently confirmed after isolation of the pathogen from relevant clinical samples [49] . SeptiFastpositive/culture-negative results could conceivably come from nonviable organisms in the blood (resulting from ongoing antibiotic treatment), cell-free DNA released from infected or colonized remote infection sites, or antibiotic interference with culture. Thus, evaluation of molecular diagnosis of sepsis requires a reference method based on multiple data types since blood culture does not detect many true sepsis cases [3] . On the other hand, culture consistently identified some organisms that were not identified by SeptiFast, possibly due to the larger volume of blood analyzed by culture and the lower limit of SeptiFast detection of approximately 3–30 CFU/ml [38] . In addition, some organisms that cause sepsis are not detected by the SeptiFast method. Nevertheless, SeptiFast and blood culture results were usually in agreement, suggesting that SeptiFast can add value as an adjunct to blood culture by both identifying pathogens not identified by blood culture and by identifying pathogens more rapidly than blood culture. SeptiFast-negative/ culture-negative specimens from patients deemed to be infected based on clinical observations or other molecular markers could result from unculturable organisms, since SeptiFast identifies only culturable organisms. Expert Rev. Mol. Diagn. 10(4), (2010)

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PCR/ESI-MS for rapid diagnosis of bloodstream infections

The time required to conduct a SeptiFast ana­lysis is less than 6 h; however, the time to the final results in real clinical settings may be significantly longer. In a study that modeled a one batch per day model, the median time for a SeptiFast result was 27 h; it was 18 h for the two batch per day model [40] . Thus, the practical considerations of specimen transport, batching specimens for testing and result reporting increase the time from the theoretical minimum turn-around time substantially. Nevertheless, even at 27 h, the SeptiFast results are available more rapidly than bloodculture results, and SeptiFast provides a measurable amount of ‘gainable days’ of adequate antimicrobial therapy [41] . This combination of shorter times to microbe identification and detection of organisms missed by culture proves the value of molecular methods. These pioneering SeptiFast studies were the first significant commercial attempts to use molecular methods to identify the organisms present in patients with bloodstream infections, and these studies have paved the way for future molecular methods and have established the benchmarks by which the value of newer molecular methods can be assessed. SeptiTest® & LOOXSTER® /VYOO™

An important consideration in molecular ana­lysis of bloodstream infections is the fact that blood contains human DNA located in circulating white blood cells. When whole blood is treated to extract and purify pathogen DNA, human DNA is co-isolated in great excess relative to pathogen DNA. The presence of excess human DNA is inhibitory to PCR and decreases the sensitivity of pathogen detection in bloodstream infections [50–52] . Although PCR sensitivity could theoretically be increased by extracting DNA from larger volumes of blood, the co-extraction of increasingly proportionate amounts of human DNA results in diminishing returns. The MolZym product known as SeptiTest® incorporates a unique sample-preparation methodology that reduces the burden of human DNA in an extracted blood sample. The method employs gentle lysis of white blood cells followed by DNase treatment to remove human DNA. This is followed by lysis of bacterial and fungal cells under more severe conditions. Thus, pathogen DNA is recovered in the presence of a greatly reduced burden of contaminating human DNA. PCR amplification of 16S ribosomal DNA and sequencing are used to identify the micro­organisms present in the sample. In theory, the MolZym sample preparation method can be used with any back-end molecular test. In analytical studies with spiked specimens, the sensitivity of SeptiTest was reportedly increased up to 1000-fold for spiked blood specimens relative to conventional spin columnbased technology with a lower limit of detection of 50 CFU/ml of blood [53–56] . A potential disadvantage of this approach is that freely circulating DNA in the blood from pathogens released from remote site infections is digested along with the human white cell DNA. Likewise, intracellular pathogens with easy to lyse membranes might be lost. The relative quantities of these sources of DNA in bloodstream infections are not known. The SIRS-Lab product LOOXSTER® uses a strategy that exploits the methylation differences between bacterial/fungal DNA and human DNA to enrich the clinical sample in pathogen www.expert-reviews.com

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DNA by affinity chromatography followed by 16S rDNA gene amplification using a product known as VYOO™ [57] . In analytical studies, the approach resulted in substantial alteration of the pathogen–human DNA ratio. Approximately 90% of eukaryotic DNA was removed. This significantly decreased the signal loss in amplification due to human DNA with sensitivity elevated at least tenfold compared samples not subjected to pathogen DNA enrichment. Early studies with small numbers of human clinical specimens are also beginning to show promising results for this approach [41,57] . PCR/electrospray ionization/MS for bloodstream infections

A new strategy for the molecular detection of bloodstream infections couples broad-range PCR amplification to electrospray ionization/MS (PCR/ESI-MS). Previous versions of this method, invented by Ibis Biosciences, now a part of Abbott Molecular, were known commercially as TIGER [58] or the Ibis T5000 [59] . The current commercial hardware platform that conducts the MS ana­lysis is now known as the Abbott PLEX-ID and the assay for direct ana­lysis of bloodstream infections that runs on the PLEX-ID is called the BAC Spectrum Assay. This technique uses primers designed to genomic regions highly conserved regions across the bacterial or fungal domains of life. The method was initially developed for the identification of microbes, including previously unknown or unculturable organisms in samples where multiple microbes may be present, primarily for biodefense applications [58,60,61] . It is now being developed for diagnosis of bloodstream infections. The steps in the PCR/ESI-MS process are illustrated in Figure 1 and the PCR/ESI-MS assay is compared with other methods in Table 2 . Briefly, multiple pairs of primers are used to amplify carefully selected regions of bacterial or fungal genomes; the primer target sites are broadly conserved but the amplified region carries information on the microbe’s identity in its nucleotide base composition. Regions like this appear in the DNA that encodes ribosomal RNA and in housekeeping genes that encode essential proteins. Following PCR amplification, a fully automated ESI MS ana­lysis is performed on the PCR/ESI-MS instrument. The mass spectrometer effectively weighs the PCR amplicons, or the mixture of amplicons, with sufficient mass accuracy that the composition of A, G, C and T can be deduced for each amplicon present. The base compositions are compared with a database of calculated base compositions derived from the sequences of known organisms and to signatures from reference standards previously determined via PCR/ESI-MS (Figure 1) . Although not as information rich as the sequence (the linking order of A, G, C and T bases is not determined using ESI-MS), for diagnostic purposes the nucleotide composition of a nucleic acid has the same practical value. For example, in the PLEX-ID BAC Spectrum assay, eight PCR reactions yield sufficient information in the base compositions of the amplicons to identify most bacteria to the species level. A critical advantage of the PCR/ESI-MS technology is that a probe is not required. When designing a probe, the target nucleic acid sequence must be known. The difference 403

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Ecker, Sampath, Li et al.

of analyzing multiple amplification products in a fast and automated fashion. With the PLEX-ID instrument, an amplification reaction can be analyzed in the mass spectrometer in 30 s, enabling ana­lysis of approximately 30 microtiter plates in a 24‑h Conserved Variable Conserved Broad-range PCR Broad-range period. This high-throughput capability DNA DNA DNA primer primer allows each specimen to be interrogated with multiple PCR reactions to increase both the breadth of coverage and the resolving power, allowing differentiation of closely related species. Measure nucleic acids Identify the organisms STEP STEP Highly precise mass Base composition fingerprints The BAC Spectrum assay for bloodstream 3 4 spectrometry Electron infections analyzes the bacterial domain of multiplier life with eight amplification reactions and ESI needle nine primer pairs (two primer pairs are A: 17 multiplexed) and combines the information Capillary G: 30 Ion from all reactions to identify the bacteria C: 11 store T: 61 Sample present. Of the nine primer pairs, three are targeted to different regions of 16S riboReflection somal DNA, one is targeted to 23S ribosomal DNA, and the remaining four are Figure 1. Identification and genotyping of microbes by PCR/ESI/mass targeted to genes encoding universally conspectrometry. ESI: Electrospray ionization. served housekeeping genes tufB, rplB, valS and rpoB (two regions). The coverage of the between the PCR/ESI-MS approach and probe-based technolo- bacterial domain of life by the PCR/ESI-MS assay is illustrated gies is that the mass spectrometer does not anticipate the ampli- in Figure 2 . Primers targeting housekeeping genes are important fication product being measured. The amplicon is weighed, the for two reasons. First, it is frequently the case that the informabase composition fingerprint that corresponds to the weight is tion needed to distinguish closely related species is not contained calculated, and the resulting finger­print is compared with those within the ribosomal DNA sequence, so the mass spectral signaof all known organisms in a database. In the event that there is no tures obtained from these housekeeping gene targets supplements match of the measured base composition with a sequence in the the information obtained from the ribosomal targets. The ability database, the nearest neighbor organism is identified in a manner to distinguish species of bacteria by integrating information from similar to that used in identification of related organisms using multiple signatures has been described previously [59] . Second, the sequence data, such as the Blast search algorithm. dynamic range of amplification of mixed populations of microbes As with broad PCR followed by sequencing, use of the is enhanced by using primer pairs that have nonoverlapping coverPCR/ESI-MS is equivalent to running many thousands of age. For example, when the ribosomal primer pairs amplify mixed specific tests, including tests that have not yet been developed, populations of microbes, the lower abundance representatives are because the identity of the infectious organism does not need detected at reduced sensitivity owing to competitive amplification to be anticipated. The PCR/ESI-MS platform not only identi- of the higher abundance species. When primer pairs that do not fies organisms present in a clinical sample but is also capable of overlap in coverage are included in the ana­lysis, like those to the providing information about the microbe, such as its strain type, housekeeping genes, this competition is avoided. This is exempliwhether or not it contains genes that mediate resistance to certain fied later in a section describing detection of mixtures. drugs and whether it carries certain virulence factors [62,63] . In addition to bacterial identification, presence of three imporIn any nucleic acid-based microbial identification schema, tant drug-resistance markers are simultaneously interrogated by whether it be based on amplification followed by probes, sequenc- the assay. The first is mecA, which is a molecular determinant of ing or MS, it is essential to analyze the regions of the microbial antibiotic resistance to b-lactam antibiotics [64] . The second are genome that contain the information needed for identification of the vanA and vanB genes, which encode resistance to vancomyimportant bacterial and fungal species. 16S ribosomal DNA has cin [65] . Finally, the blaKPC gene, which encodes resistance to the been widely used for microbial identification. However, not all carbapenem class of antibiotics in Klebsiella pneumoniae and other regions of the 16S ribosomal DNA are equivalent – not all provide Gram-negative bacilli [66] , is interrogated. a universal amplification target and not all have useful information The presence of Candida DNA is determined with three primer content. It is, therefore, advantageous to amplify and analyze mul- pairs targeting ribosomal DNA that can identify and differentiate tiple target sites within the ribosomal DNA. A major strength of members of Candida spp., including Candida albicans, Candida the PCR/ESI-MS method is that the mass spectrometer is capable glabrata, Candida tropicalis, Candida parapsilosis, Candida

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STEP Identify genomic regions for identification 1 Variable DNA sequences flanked by conserved regions

404

STEP Amplify nucleic acids Use broad-range, unbiased 2 PCR primers

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Bacillus subtilis Brevibacillus brevis

Alloiococcus otitis Aerococcus urinae Aerococcus sanguinicola Abiotrophia defectiva Weissella confusa Leuc. pseudomesenteroides Leuconostoc lactis Leuconostoc mesenteroides Facklamia hominis Facklamia sourekii Lactobacillus rhamnosus Lactobacillus casei

]

Clostridiales

Clostridium difficile Clostridium sordellii Clostridium spp. Clostridium novyi C. clostridioforme C. bifermentans C. perfringens C. innocuum

S. pneumoniae S. porcinus S. ratti S. salivarius S. sanguinis S. sobrinus S. uberis S. mutans S. macacae

Mycoplasma pneumoniae

Erysipelothrix rhusiopathiae

CM dec 16 2009

Fusobacteria

Firmicutes

NotÊ348

NotÊ346

Francisella philomiragia Francisella tularensis

NotÊ349

Coxiella burnetti

Legionella pneumophila Legionella longbeachae

Grimontia hollisae Vibrio paraheamolyticus Vibrio vulnificus Vibrio fluvialis Vibrio cholerae [ 358 ] Vibrio mimicus

Aeromonas hydrophila Aeromonas spp. [ 3921 ] Aeromonas schubertii Shewanella putrefaciens Anaerobiospirillum succiniproducens

Haemophilus influenzae Haemophilus parahaemolyticus Haemophilus parainfluenzae Pasteurella multocida Aggregatibacter aphrophilus A. actinomycetemcomitans

Stenotrophomonas maltophilia

Cardiobacterium hominis

Gammaproteobacteria

[ 3350 ]

Epsilonproteobacteria

Proteobacteria

[ 3346 ]

Chlamydia

Chlamydophila pneumoniae Chlamydia trachomatis

NotÊ349

Streptobacillus moniliformis Leptotrichia spp. Fusobacterium nucleatum F. necrophorum F. mortiferum F. varium

Anaerococcus prevotii Eubacterium limosum Finegoldia magna Veillonella spp. Clostridium ramosum Peptoniphilus asaccharolyticus Peptostreptococcus anaerobius

S.equi Streptococcus pyogenes S. acidominimus S. equinus S. gordonii S. agalactiae S. hyointestinalis S. alactolyticus S. intermedius S. anginosus S. vestibularis S. constellatus S. suis S. criceti S. oralis S. cristatus S. parasanguinis S. dysgalactiae

Lactobacillales

Listeria grayi Gemella haemolysans Gemella morbillorum Lysinibacillus sphaericus Paenibacillus alvei Bacillus cereus B. cereus group B. licheniformis B. megaterium B. coagulans [ 3350 ] B. circulans B. pumilis Bacillales

Kytococcus sedentarius Micrococcus lylae

Dermacoccus nishinomiyaensis Actinomyces odontolyticus

Bifidobacterium longum

Gardnerella vaginalis

Actinobacteria

Treponema pallidum Borrelia burgdorferi B. afzelii B. hermsii B. garinii B. turicatae

Spirochaetes

Sphingobacterium multivorum

Bacteroidetes

Capnocytophaga canimorsus Capnocytophaga cynodegmi Chryseobacterium indologenes Elizabethkingia meningoseptica Riemerella anatipestifer

Campylobacter jejuni Campylobacter gracilis Campylobacter coli Campylobacter fetus Arcobacter butzleri

Escherichia coli E. spp. E. hermannii E. vulneris E. fergusonii Shigella flexneri Klebsiella pneumoniae Klebsiella oxytoca [4675] Morganella morganii Pantoea agglomerans Photorhabdus luminescens Plesiomonas shigelloides Leclercia adecarboxylata Yokenella regensburgei Yersinia pseudotuberculosis Y. pestis Y. enterocolitica Raoultella spp. Providencia alcalifaciens Providencia rettgeri Ewingella americana Hafnia alvei Providencia stuartii

Pseudomonas alcaligenes Pseudomonas fluorescens Pseudomonas mendocina Pseudomonas putida Pseudomonas stutzeri Pseudomonas oryzihabitans Psychrobacter immobilis Pseudomonas luteola Pseudomonas aeruginosa

Bartonella henselae Bartonella quintana [ 3921 ] Brucella spp.

Cedecea neteri Citrobacter amalonaticus C. koseri C. freundii Cronobacter sakazakii Edwarsiella hoshinae Edwarsiella tarda Enterobacter aerogenes Enterobacter cloacae E. asburiae E. cancerogenus E. gergoviae E. hormaechei Kluyvera cryocrescens K. ascorbata K. intermedia Tatumella ptyseos Rahnella aquatilis Salmonella enterica [ 358 ] Proteus penneri P. vulgaris P. mirabilis Serratia marcescens [ 3921 ] Serratia liquefaciens S. rubidaea S. plymuthica

Acinetobacter baumannii Acinetobacer haemolyticus Acinetobacter calcoaceticus Acinetobacter lwoffii Acinetobacter johnsonii Moraxella catarrhalis Moraxella nonliquefaciens Moraxella lincolnii [ 3921 ] Moraxella osloensis

Burkholderia mallei Burkholderia pseudomallei Burkholderia cepacia Burkholderia fungorum Achromobacter denitrificans Achromobacter xylosoxidans Alcaligenes faecalis Bordetella bronchiseptica Bordetella holmesii [ 358 ] Bordetella pertussis Cupriavidus pauculus Delftia acidovorans Oligella ureolytica [ 3350 ] Ralstonia picketti

Brevundimonas diminuta Brevundimonas vesicularis

Sphingomonas paucimobilis

Methylobacterium zatmanii Methylobacterium mesophilicum Ochrobactrum anthropi Agrobacterium tumafaciens

Chromobacterium violaceum Neisseria meningitidis Neisseria gonorrhoeae N. cinerea N. mucosa N. flavescens N. sicca N. lactamica N. subflave Kingella kingae [ 3350 ]

Betaproteobacteria

[ 3346 ]

Alphaproteobacteria

Roseomonas cervicalis Roseomonas mucosa Roseomonas gilardii

Rickettsia rickettsii Rickettsia felis Rickettsia canadensis Ehrlichia chaffeensis

Anaplasma phagocytophilum Rickettsia typhi

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.

Lactococcus garvieae Lactococcus lactis Enterococcus faecalis Enterococcus avium Enterococcus cecorum Enterococcus casseliflavus Enterococcus spp. E. faecium [ 3767 ] E. gallinarum [ 3768 ] E. durans Globicatella sanguinis

[ 3350 ]

Staphylococcus aureus S. arlettae S. auricularis S. capitis S. epidermidis S. cohnii S. haemolyticus S. delphini S. intermedius S. equorum S. lugdunensis S. gallinarum S. saccharolyticus S. homonis S. saprophyticus S. kloosii S. schleiferi S. lentus S. simulans S. sciuri S. vitulinus S. xylosus S. warneri [ 879

2249 ]

C. argentoratense C. minutissimum [ 3346 ] [ 3346 ] C. propinquum C. glucuronolyticum C. pseudodiphtheriticum C. urealyticum

Corynebacterium diphteriae C. striatum C. amycolatum C. kutscheri C. jeikeium C. afermentans C. coyleae C. renale C. accolens C. bovis C. auris C. macginleyi C. xerosis

Listeria innocua [ Listeria monocytogenes Macrococcus caseolyticus

Microbacterium sp. Micrococcus luteus Mycobacterium chelonae Mycobacterium fortuitum Mycobacterium neoaurum Mycobacterium simiae Mycobacterium spp. Nocardia spp. Oerskovia turbata Propionibacterium acnes Rhodococcus equi Rothia dentocariosa Rothia mucilaginosa Tsukamurella inchonensis Tsukamurella strandjordii

Odoribacter splanchnicus Prevotella oralis Porphyromonas gingivalis Parabacteroides distasonis Bacteroides thetaiotaomicron B. caccae B. ovatus B. fragilis B. capillosus B. ureolyticus B. vulgatus

Arcanobacterium bernadiae Arcanobacterium haemolyticum Arcanobacterium pyogenes Actinomyces neuii [ 3921 ] Actinomyces israelii Actinomyces radingae Actinomyces turicensis Actinomyces viscosus Arthrobacter agilis Brevibacterium casei Brevibacterium paucivorans Dermabacter hominis Gordonia terrae Kocuria kristinae Leifsonia aquatica

NotÊ349

[ 346, 348, 349, 361 ] Ribosomal primer pairs with broad bacterial coverage unless indicated otherwise

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PCR/ESI-MS for rapid diagnosis of bloodstream infections

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lusitaniae, Candida guilliermondii, Candida dubliniensis and Candida krusei. These broad-spectrum primer pairs were selected by careful alignment of fungal and human rRNA gene sequences and were designed to target regions where the 3´ ends of primers hybridize to regions substantially different from the human rRNA gene sequence. In addition, the assay includes a primer pair that targets the mitochondrial small subunit ribosomal RNA gene of the C. albicans group of Candida spp. and provides confirmation of and speciation of C. albicans, C. tropicalis, C.  arapsilosis, C. dubliniensis, C. guilliermondii and Candida kefyr. Triangulation of base composition ana­lysis of multiple targets for microbial identification

In contrast to sequencing, base composition ana­lyses and its ability to distinguish microbial species is not intuitive. With the PCR/ESI-MS method, the base composition, rather than the sequence, of the amplicon is determined and compared with a database of calculated base compositions derived from a curated database of sequences. Thus, the order of the nucleotides is not known. Obviously the base composition is less information-rich than the sequence. The PCR/ESI-MS method makes up for the lower information content of base composition compared with sequencing by amplifying multiple regions of the microbial genome instead of one and by using the aggregate information derived from these PCR reactions to identify the microbe. Both sequencing and base composition ana­lysis share the common feature of conducting a comparison with a curated database of sequences and the conventions established for curation of databases used for ana­lysis of sequence are directly applicable to the data obtained using PCR/ESI-MS. The Clinical Laboratory Standards Institute recently published approved guidelines for the identification of bacteria and fungi by DNA target sequencing [67] . These guidelines address the issues of the quality of the sequences used in the database, sequence redundancy, periodic updates, organism nomenclature errors and other issues of concern when using a database comparison to identify organisms. The PCR/ESI-MS database is curated in accordance with these guidelines. The PCR/ESI-MS database was generated and is maintained and kept up-to-date as follows. For each primer pair in the assay, a reference sequence for the target gene from the best available published reference sequence is chosen. Additional sequences of the target gene are aligned to the reference sequence. Using the primer sequences for the selected assay, the bacterial section of GenBank is analyzed computationally for entries that would be amplified by the primer pairs. The resulting sequences are curated using bioinformatic tools by comparison with the reference sequence and the expected base compositions for the target region are calculated and used to populate the database. These computationally generated base compositions are augmented with experimentally measured base compositions from reference isolates of the target organisms that have been certified based on established methods. Experimentally observed base compositions from each primer pair target region are compared with the PCR/ESI-MS database of base compositions. The information on base counts from each primer pair provides a signature for the organism. 406

A visualization of the ability of base composition to identify and speciate Candida is shown in Figure 3A . The columns represent the four amplification target regions for the three chromosomal and the mitochondrial rDNA targets. Each row of four adjacent colored cells represents the base composition signature of each Candida spp. Within each column, cells are color-coded to represent with similar hue signatures that are close to each other in base composition. The intensity of the color in each cell was computed to visually reflect the distinctiveness of its signature, with pale or pastel colors indicating base compositions closer to the overall consensus and more saturated colors representing the most divergent base counts. Although there is occasional overlap of base compositions where two or more Candida spp. might have the same base composition in one target region, the use of multiple target regions and triangulation of the signatures assures correct speciation, yet allows for expected biological variation, such as mutations resulting in differences in two isolates of the same species. As with a sequence similarity search, ana­lysis of base compositions enables identification of variants from the database based on the closest match. A visualization of the base composition signatures obtained for 332 phylogenetically diverse bacteria in the PCR/ESI-MS database using the same color-coding schema as in Figure 3B is shown in a heat-map format in Figure 4 . These data show the near universal primer coverage of the 16S rDNA primers (first three columns). Although no rDNA primer is completely universal (e.g., the Fusobacteria are not amplified by BCT346 and the Actinomycetales are not amplified by primer pair BCT349), the combination of all nine targets provides universal coverage of the bacterial domain of life. Several of the primer pairs provide specialty functions. For example BCT2249, targeted to the tufB gene, was designed to speciate the Staphylococcaceae, where S. aureus is unambiguously distinguished from the other Staphylococcus spp., but does not prime outside of this genus [63] . Similarly BCT358 (targeting valS) provides high resolving power of the Enterobacteriaceae. Detection of mixed populations of bacteria in direct blood specimens

A significant number of bloodstream infections are polymicrobial. The PCR/ESI-MS method can identify mixed populations of bacteria as this technique directly analyzes nucleic acids extracted from blood. A representative result from a blood specimen from a patient infected with both S. aureus and Pseudomonas aeruginosa is shown in Figure 5. Analysis of individual spectra derived from amplification from the Gram-positive-centric primers (top two spectra) clearly show base compositions that match S. aureus. Spectra from the Gram-negative-centric primers, such as BCT3346 (third spectrum), show base compositions consistent with P. aeruginosa. However, the broad rDNA primers, such as BCT361 and BCT346 (fourth and fifth spectra), show signals and base compositions that correspond to both S. aureus and P. aeruginosa in an approximately 3:1 ratio. In addition, signals from BCT879, targeting the mecA gene are clearly present (bottom spectrum), indicating that the S. aureus present harbor methi­ cillin resistance. Thus, the combination of signals from this blood Expert Rev. Mol. Diagn. 10(4), (2010)

PCR/ESI-MS for rapid diagnosis of bloodstream infections

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A

FUN3030 25S rDNA

Organism

FUN3031 25S rDNA

Technology Report

FUN3766 25S rDNA

FUN3865 mitochond. rDNA

Candida albicans

A30 G38 C24 T36

A36 G44 C24 T34

A36 G39 C31 T42

A32 G23 C22 T38

Candida colliculosa

A31 G38 C23 T36

A37 G43 C25 T35

A40 G33 C29 T52

A38 G20 C14 T43

Candida cylindracea

A35 G35 C23 T35

A36 G42 C23 T35

A37 G37 C28 T51

No prime

Candida dubliniensis

A30 G38 C24 T36

A36 G44 C23 T35

A36 G39 C29 T45

A26 G27 C26 T32

Candida famata

A33 G37 C22 T36

A38 G40 C23 T37

A37 G37 C30 T49

A36 G16 C17 T24

Candida fennica

A31 G36 C24 T31

A36 G42 C24 T36

A33 G39 C32 T44

A38 G19 C18 T51

Candida glabrata

A32 G36 C24 T36

A34 G45 C28 T37

A37 G36 C30 T51

A40 G19 C13 T48

Candida guilliermondii

A31 G37 C22 T38

A34 G41 C23 T40

No prime

A35 G16 C19 T23

Candida kefyr

A32 G37 C24 T35

A36 G43 C25 T34

A39 G35 C30 T49

No prime

Candida krusei

A31 G43 C22 T29

A36 G48 C31 T34

A33 G43 C31 T44

No prime

Candida lusitaniae

A38 G42 C27 T26

A37 G36 C30 T51

No prime

A37 G43 C26 T33

A33 G41 C31 T46

No prime

Candida parapsilosis

A32 G36 C24 T36 A32 G49 C25 T22 A33 G48 C25 T22 A32 G36 C21 T39

A37 G40 C25 T38

A37 G39 C28 T45

A30 G20 C19 T43

Candida rugosa

A26 G41 C23 T26

A38 G42 C25 T35

A37 G40 C30 T46

A33 G20 C17 T39

Candida norvengensis

Candida sp. (e.g., C. rugosa)

A36 G32 C19 T25

A38 G42 C25 T35

A37 G38 C31 T48

No prime

Candida sphaerica

A33 G36 C24 T35

A36 G43 C25 T34

A39 G35 C30 T49

A34 G20 C17 T45

Candida tropicalis

A32 G36 C21 T39

A34 G44 C25 T35

A37 G38 C28 T46

A38 G28 C24 T41

B

A+C

Max length C+T

A+T

G+C

Consensus length

A+G Min length G+T C+M+Y components

K component

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.

specimen analyzed without culture indicates an infection that requires antimicrobial therapy that covers both these organisms, including consideration of drug resistance. Simultaneous detection of culturable & unculturable organisms

Our view of organisms that cause bloodstream infections is biased because culture is the only method widely used. Since blood cultures are reported to be negative in more than 50% of www.expert-reviews.com

the cases where true bacterial or fungal sepsis is believed to exist [3] , there is a missing component to our understanding of infections. The PCR/ESI-MS method detects pathogens with no bias due to culturability. Aerobic, anaerobic, culturable, fastidious and unculturable organisms are identified in the same way. To get a perspective on the fractional occurrence of culturable organisms, we surveyed samples from seven hospital micro­biology laboratories in Cleveland (OH, USA), Nashville (TN, USA), Tuscon (AZ, USA), New York (NY, USA), Baltimore (MD, 407

Technology Report

Phylum

Ecker, Sampath, Li et al.

Order

BCT346 16S rRNA

BCT348 BCT361 BCT349 16S rRNA 16S rRNA 23S rRNA

BCT3350 rplB

BCT2249 tufB

BCT358 valS

BCT3346 rpoB

BCT3921 rpoB

Bacillales

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Firmicutes

Staphylococcaceae

Lactobacillales

Bacteroidetes

Actinobacteria

Clostridiales

Actinomycetales

Bacteriodales Flavobacteriales

Alphaproteobacteria

Rhizobiales

Betaproteobacteria

Spirochaetes

Burkholderiales

Rickettsiales

Nesseriales

Gammaproteobacteria

Enterobacteriales

Pseudomonoadales Aeromonadales Pasteurellales Vibrionales

Epsilonproteobacteria Chlamydia Fusobacteria

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.

USA), Salt Lake City (UT, USA) and Barcelona (Spain). To create a metric comparable among the hospitals due to different bed numbers, the positive blood culture counts for each organism category at each site were converted to a percentage of the total blood culture results reported for that hospital. The mean 408

percentages across the seven sites are plotted in Figure 6 with the error bars representing one standard deviation from the mean. Reported organism classes making up less than 1% on average across the sites were binned together. Owing to differences in the reporting precision among various hospital microbiology Expert Rev. Mol. Diagn. 10(4), (2010)

PCR/ESI-MS for rapid diagnosis of bloodstream infections

Technology Report

fluid from patients clinically suspected of infection by Ehrlichia or other tick-borne S. aureus BCT3350 disease organisms [68] . One of the challenges of evaluating new technology that 22,000 24,000 goes beyond the currently available methods is identifying a reference method to comA43 G28 C19 T35 pare the results. A combination of culture, S. aureus BCT2249 antibody-based methods, and broad-range 35,000 37,000 PCR amplification followed by hybridization to specific probes were used in combination to provide a basis for comparison A21 G35 C32 T24 P. aeruginosa BCT3346 of PCR/ESI-MS results. A two-step, colorimetric microtiter plate PCR assay capable 34,000 36,000 of detecting and differentiating medically A27 G33 C29 T20 important Ehrlichia species was used [69,70] . S. aureus A29 G30 C25 T24 The first step involved PCR amplification BCT361 P. aeruginosa of a genus-specific region in the 16S rRNA 32,000 34,000 gene, which has been described elsewhere [71,72] . In the second step, the PCR ampliA30 G31 C23 T15 S. aureus fication product was identified and differA27 G30 C21 T21 BCT346 P. aeruginosa entiated to Ehrlichia chaffeensis, Ehrlichia 29,000 31,000 ewingii or Anaplasma phagocytophilum using three species-specific probes. The same PCR A22 G11 C21 T21 enzyme immunoassay (PCR-EIA) format BCT879 mecA gene and procedure was used to detect Rickettsia rickettsii by targeting a 154-bp fragment, as 22,000 24,000 Mass (Da) described previously [73] . An overview of the results from this study Figure 5. Spectra of a sample from a patient with a mixed infection. A blood is shown in Figure 7. Of 213 specimens anaspecimen from a patient co-infected with Staphylococcus aureus containing a mecA lyzed, the majority of detected infections gene and Pseudomonas aeruginosa as analyzed by PCR/electrospray ionization/mass spectrometry. Individual spectra from six of the nine bacteria-targeted amplifications are in this patient population were E. chaffeenshown. M/Z was converted to the M domain and the scales were selected to visualize all sis, but other Ehrlichia and Rickettsia spp. detected peaks. were also identified, consistent with the hypothesis that this patient population is laboratories (some laboratories report certain organisms at the disproportionately infected with unculturable organisms [68] . species level, while others report at the genus level), the data In comparison to the PCR-EIA, the PCR/ESI-MS sensitivity, in Figure 6 are reported based on the highest precision reported specificity, and positive- and negative-predictive values were 95.0, from all seven hospital laboratories. These combined percent- 98.8, 95.0 and 98.8%, respectively. The 38 specimens that were ages are displayed by phylum and abundance. Overall, there positive for Ehrlichia by both PCR/ESI-MS and PCR-EIA were were 138 unique species identified by culture methods at the further characterized to the species level, with 100% agreement seven hospitals. The distribution of pathogens is highly biased in between the two assays. In addition, R. rickettsii was detected favor of approximately 30 of the most common pathogens, which by PCR/ESI-MS from four specimens, which were confirmed was fairly consistent across all seven hospitals, as shown by the retro­spectively by serology and PCR-EIA. In three specimens, the standard deviations. These data provide a benchmark of cultur- PCR/ESI-MS assay identified P. aeruginosa, Neisseria meningitiable organisms, which will be compared with the PCR/ESI-MS dis, and S. aureus; these identifications were confirmed by culture ana­lysis of direct patient specimens in the future. and/or clinical diagnosis as being clinically relevant (Table 3) . Different patient populations would be expected to vary in the frequency of unculturable organisms. Febrile patients who reported The PCR/ESI-MS method is quantitative recent tick bites or travel in the wilderness are more likely than An important aspect of PCR/ESI-MS is that the method is quanother patient populations to be infected with unculturable organ- titative [58,59,61] . As discussed earlier, culture methods are generally isms, such as Ehrlichia, Rickettsia, Bartonella, Anaplasma or other not quantitative and little is known regarding the levels of pathounculturable Proteobacteria, or Spirochaetes, such as Borrelia spp. gens in bloodstream infections. Even less is known about levels of However, these patients could also be infected with more common pathogen nucleic acids in bloodstream infections and there is potenpathogens. To investigate pathogens infecting this patient popula- tial diagnostic value in a quantitative measurement. In each PCR/ tion we used PCR/ESI-MS on whole blood, plasma or cerebrospinal ESI-MS experiment, an internal calibrant is added to every well of

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A16 G23 C21 T19

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409

Technology Report

Ecker, Sampath, Li et al.

the assay plates. This calibrant acts as a PCR control and a standard for quantification in each well. Essentially, the peak heights in the mass spectrum for amplicons and calibrant are use to determine the relative ratios of calibrant to microbe. As the original concentration of calibrant was known, the concentration of pathogen,

measured in genome copy number per milliliters of specimen, can be determined accurately. In experiments with 37 samples that tested positive for Ehrlichia nucleic acid concentrations ranged from near the lower limit of detection to over 500,000, with an average copy number of near 100,000/ml. As described earlier,

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8 other fungi Candida guilliermondii Candida krusei

Candida species

Candida tropicalis Candida parapsilosis Candida glabrata Candida albicans Fusobacteria 10 other Bacteroidetes

Bacteroidetes

Bacteroides fragilis Bacteroides spp. 12 other Actinobacteria Microbacterium spp. Micrococcus luteus Microcococcus spp.

Actinobacteria

Propionibacterium spp. Diptheroids Corynebacterium spp. 61 other Proteobacteria Citrobacter spp. Acinetobacter spp. Enterobacter spp. Acinetobacter lwoffii Citrobacter freundii Morganella morganii Salmonella spp. Pseudomonas spp. Haemophilus influenzae

Proteobacteria (Gram-negatives)

Enterobacter aerogenes Stenotrophomonas maltophilia Klebsiella oxytoca Proteus mirabilis Serratia marcescens Acinetobacter baumannii Enterobacter cloacae Pseudomonas aeruginosa Klebsiella pneumoniae Escherichia coli 11 other Firmicutes Bacillus cereus Clostridium perfringens Staphylococcus lugdunensis Lactobacillus spp.

Firmicutes (Gram-positives)

Clostridium spp. Streptococcus pyogenes Streptococcus agalactiae Enterococcus spp. Staphylococcus haemolyticus Bacillus spp. Streptococcus pneumoniae Enterococcus faecalis Enterococcus faecium Streptococcus spp. Staphylococcus aureus CoNS

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

30% 40%

Figure 6. Frequency of organisms identified by the microbiology laboratory from seven hospitals. CoNS: Coagulase-negative staphylococci.

410

Expert Rev. Mol. Diagn. 10(4), (2010)

PCR/ESI-MS for rapid diagnosis of bloodstream infections

measurement of CFU per milliliter in bloodstream infections for common culturable organisms ranges from very low (~10 CFU/ml) to very high, especially in pediatric patients and newborns [7] . A series of clinical trials have been arranged to evaluate PCR/ESI-MS based quantitative measures for a broad variety of pathogens and clinical relevance.

Technology Report

E. ewingii 3 E. chaffeensis 37

R. rickettsii 4 B. vulgatus 1 N. meningitidis 1 P. aeruginosa 1

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Clinical utility of the PCR/ESI-MS instrument

The PCR/ESI-MS system is available commercially and systems are currently in use for research and disease surveillance at 20 sites in the USA and Europe. With the current system, all steps, including sample collection and preparation, and the automated PCR, MS ana­lysis, signal processing and report generation, can be carried out by clinical laboratory personnel within a 4–6-h period. The mass spectrometer provides data in approximately 30 s, uses no consumable products other than solvents, can analyze approximately 3000 PCR reactions in 24 h in a completely automated fashion. No training in MS is required for personnel who operate the instrument or for those who interpret the results. Limitations & future improvements

Although broad-range PCR ana­lysis of samples has many advantages, it comes with the difficulty of requiring exquisitely clean reagents that are free from adventitious bacterial and fungal DNA along with work flow-processes that prevent introduction of contamination in all steps leading up to PCR. Efforts focused on this issue have resulted in reliable manufacturing processes and quality-control procedures that successfully avoid contamination. Virtually all commercially obtained materials contain small amounts of bacterial genomic material [74] . This material is introduced even under the most stringent of conditions either

No detection 165

S. aureus 1

Figure 7. PCR/electrospray ionization/mass spectrometry ana­lysis of samples from patients with suspected tickborne 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.

by the handling of materials during early production stages or by the contact of the materials with air or surfaces during production. Industrial processes were developed for the production of DNA-free reagents. This includes the production of reagents for PCR, perhaps the most critical reagents from a contamination point of view. Key to the production of contaminant-free reagents was the development of a closed-loop system that allows the ‘cleaning’ of reagents after mixing and then filling of custom intravenous bags for the storage of those reagents. The cleaning of the reagents includes passing them through a 0.2‑µm filter to remove organisms, as well as through a special filter designed to remove nucleic acid contaminants. The final kit format for

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 Serum rickettsial IgG titer of Rocky Mountain Spotted Fever 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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Technology Report

Ecker, Sampath, Li et al.

the current PLEX-ID hardware platform is a 96-well plate that can be used in standard liquid-handling robotics, as well as in our automated clean-up procedure. An important source of adventitious DNA is the enzymes used in the PCR reactions. Commercial procedures have been established with a commercial producer of Taq polymerase and large batches of enzyme that meet stringent specifications are regularly obtained. Only lots of enzyme that pass quality-control procedures are used. An important limitation of molecular methods is that only three genes associated with phenotypic drug resistance are currently interrogated by the BAC Spectrum assay. The current assay is limited to mecA, which is a molecular determinant of antibiotic resistance to b-lactam antibiotics [64] , the vanA and vanB genes, which encode resistance to vancomycin [65] , and the bla KPC gene, which encodes resistance to the carbapenem class of antibiotics in Klebsiella pneumoniae and other Gram-negative bacilli [66] . Although this is a good start, the molecular mechanisms of resistance to other important antibiotics are either not fully understood or require ana­lysis of too many regions of the microbial genome to be included in the current assay format. Analysis of drug-resistance mechanisms is a highly active area of research and we can expect rapid increases in understanding the molecular underpinnings of resistance. Further improvements in multiplex technology will enable more drug-resistance markers to be analyzed by the PCR/ESI-MS method.

collection, lysis, extraction and enrichment of pathogen nucleic acids with a back-end molecular ana­lysis technology that identifies virtually all pathogens. Both components are essential to success, and the lack of a suitable front end has held back progress in the field. The pioneering efforts of Roche with its SeptiFast product are beginning to show proof of principle that microbial identification by direct molecular ana­lysis of bloodstream infections is achievable. Significant hurdles remain in specimen preparation, and sensitive, comprehensive and rapid ana­lysis of a broad range of pathogens. Automation will be essential to provide speed, throughput and contamination control, and to reduce the labor requirements in molecular ana­lysis of bloodstream infections. Advanced analytical techniques, such as MS, have the potential to change the current paradigm. These are now available and will be evaluated in clinical studies shortly. The new molecular technologies will also lead to significant new discoveries. For example, microbial causes of many disease states currently not believed to be of infectious origin will be identified. New pathogens will be discovered and the role of microbial infections in broader disease states will be revealed [75–77] . A benefit of determination of microbial drug susceptibility will be that antibiotic stewardship will be enhanced. It will be no longer necessary to treat patients with broad empirical therapy as the correct and optimal antimicrobial therapy can be prescribed immediately based on a rapid, comprehensive and accurate molecular test.

Expert commentary

Five-year view

During the past two decades, technical advances in molecular biology have initiated a revolution in the diagnostic microbiology field [5,6] . Molecular techniques have found their niches in laboratory diagnosis of bloodstream infections by providing rapid and highly discriminatory tools to identify the causal pathogens. The new molecular tools have the potential to improve the sensitivity of current automated blood culture systems by covering unculturable and fastidious pathogens. Methods to rapidly detect and identify pathogens responsible for bloodstream infections are desperately needed. For the most serious bloodstream infections, such as septic shock, time is of the essence as survival rates drop on an hourly basis if appropriate treatment is delayed. It is important to both identify the infecting organism and determine its sensitivities to antimicrobial agents. The current goldstandard diagnostic methods are based on culture, which is slow, labor intensive, nonquantitative and does not identify over half the cases where true bacterial or fungal sepsis is believed to exist [3] . We are now in the early stages of a new era of molecular diagnostics for bloodstream infections based on advanced technologies, such as MS, next-generation sequencing and other single-molecule detection methods. These analytical methods have the potential to identify pathogens and provide detailed subspecies information. As the molecular mechanisms for antimicrobial resistance become better understood, prediction of drug susceptibilities will be achievable by molecular methods. Specimen preparation and enrichment of pathogen DNA remains a major challenge. Successful molecular diagnostics for bloodstream infections must integrate technology for front-end specimen

The key objective of molecular diagnostics of bloodstream infections is to identify the pathogen(s) and key genetic determinants of drug resistance as rapidly as possible in order to initiate appropriate antimicrobial therapy. These objectives will be achieved within the next 5 years based on advances in the following three areas. First, specimen preparation, including collection, lysis, extraction and enrichment of pathogen nucleic acids, will undergo a quantum advance based on new approaches. The current preparative methods using columns or magnetic bead-based nucleic acid purification have not fundamentally changed in two decades, are inadequate and are not likely to advance further other than by incremental improvements. We believe that within 5 years a currently unanticipated technology will be developed to address the specimen preparation problem. In the near term, variations of the current specimen approaches will be used to successfully demonstrate the feasibility and value of revolutionary back-end analytical methods. However, the full potential of molecular methods for diagnosis of bloodstream infections will not be reached until a quantum advance in specimen preparation is achieved. Second, modern analytical methods will provide gamechanging advances in measuring the signatures in microbial DNA that simultaneously detect, identify, subspeciate and strain-type pathogen DNA. Advanced MS and next-generation single-molecule sequencing methods have the potential to analyze specimens in a massively parallel fashion and measure molecular details that will enable precise diagnosis of pathogens. Quantitative measurements will demonstrate value once the methods to measure pathogen nucleic acids are widely available.

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PCR/ESI-MS for rapid diagnosis of bloodstream infections

Third, advances in understanding the molecular nature of microbial drug resistance will provide enabling information to predict which drugs will be most effective without conventional culturebased drug-susceptibility measurements. Whole-genome sequencing methods currently under development will enable massively parallel sequencing of pathogen specimens in various stages of antimicrobial resistance development, providing detailed understanding of the complex process of drug resistance. This will enable the determination of the correct drugs based solely on molecular measurements, which is the last requirement of molecular methods that must be met before culture methods can be eliminated completely. While the automated blood culture instruments with continuous monitoring will remain the mainstay technique for detection of bacterial organisms causing bloodstream infections, molecular techniques will gradually play a dominant role in rapid identification

Technology Report

of detected bloodstream pathogens. With host responses included in the test panel, a functional identification by molecular methods can be performed routinely for virulence factor detection, antibiotic resistance determination and therapy efficacy assessment. Financial & competing interests disclosure

David J Ecker, Rangarajan Sampath, Christian Massire, Heather E Matthews, Donna Toleno, Thomas A Hall, Lawrence B Blyn, Mark W Eshoo and Steven A Hofstadler are employees of Ibis Biosciences, a subsidiary of Abbott Molecular Inc, the commercial manufacturer of the PCR/ESI-MS technology. Yi-Wei Tang is a consultant of Ibis Biosciences. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • Rapid methods for identification of pathogens responsible for bloodstream infections are in development. • The ideal test would be rapid, not require culture and would quantitatively identify all pathogens present in a blood sample. • PCR/electrospray ionization/mass spectrometry can identify pathogens without prior assumptions regarding the type of infection. • Specimen preparation and enrichment of pathogen DNA remains a major challenge.

References

7

Papers of special note have been highlighted as: • of interest •• of considerable interest

Yagupsky P, Nolte FS. Quantitative aspects of septicemia. Clin. Microbiol. Rev. 3(3), 269–279 (1990).



1

Russell JA. Management of sepsis. N. Engl. J. Med. 355(16), 1699–1713 (2006).

2

Kumar A, Roberts D, Wood KE et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34(6), 1589–1596 (2006).

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•• Current guidelines for management of patients with severe sepsis. 4

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•• Reviews the use of the PCR/electrospray ionization/mass spectrometry system for identification of bacterial, fungal and viral pathogens. Best introduction to the technology. 60

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