Rapid diagnosis of bloodstream infections with PCR followed by mass spectrometry.

PLoS One. 2013 Apr 23;8(4):e62108.

Jordana-Lluch E, Carolan HE, Giménez M, Sampath R, Ecker DJ, Quesada MD, Mòdol JM, Arméstar F, Blyn LB, Cummins LL, Ausina V, Martró E.

Microbiology Department, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain.

 

Abstract

Achieving a rapid microbiological diagnosis is crucial for decreasing morbidity and mortality of patients with a bloodstream infection, as it leads to the administration of an appropriate empiric antimicrobial therapy. Molecular methods may offer a rapid alternative to conventional microbiological diagnosis involving blood culture. In this study, the performance of a new technology that uses broad-spectrum PCR coupled with mass spectrometry (PCR/ESI-MS) was evaluated for the detection of microorganisms directly from whole blood. A total of 247 whole blood samples and paired blood cultures were prospectively obtained from 175 patients with a suspicion of sepsis. Both sample types were analyzed using the PCR/ESI-MS technology, and the results were compared with those obtained by conventional identification methods. The overall agreement between conventional methods and PCR/ESI-MS performed in blood culture aliquots was 94.2% with 96.8% sensitivity and 98.5% specificity for the molecular method. When comparing conventional methods with PCR/ESI-MS performed in whole blood specimens, the overall agreement was 77.1% with 50% sensitivity and 93.8% specificity for the molecular method. Interestingly, the PCR/ESI-MS technology led to the additional identification of 13 pathogens that were not found by conventional methods. Using the PCR/ESI-MS technology the microbiological diagnosis of bloodstream infections could be anticipated in about half of the patients in our setting, including a small but significant proportion of patients newly diagnosed. Thus, this promising technology could be very useful for the rapid diagnosis of sepsis in combination with traditional methods.

PMID: 23626775

 

Supplements:

Bacteriemia (defined as the presence of microorganisms in the blood) and sepsis (systemic inflammatory response to the inflammation caused by this infection) are life-threatening conditions that have an increasing incidence. This incidence was estimated to be 19 million cases worldwide per year (1). The incidence rate in the US in 2007 was 711.7 per 100.000 persons, well over other major diseases (Fig.1).

The administration of antibiotic therapy within the first hour of recognition of the clinical symptoms of sepsis significantly increases the probability of patient survival (2). However, this treatment should be chosen empirically, since the reference method used for the detection of pathogens in blood (blood culture followed by conventional identification methods) and for antibiotic susceptibility testing may take up to several days. Once the microbiological identification is achieved, the initial therapy can be changed, if necessary, to assure an adequate antibiotic activity against the etiologic agent, or its spectrum can be reduced to prevent antimicrobial resistance development (3). Thus, a rapid microbiological diagnosis is of paramount importance for the best outcome of the patient.

In order to accelerate the diagnostic process, it would be desirable to detect and identify the microorganisms directly from the patient’s blood overcoming the limitations of culture. Currently, molecular methods, based on the detection of bacterial nucleic acids, offer a rapid and reliable alternative to culture, reducing the time to detection and increasing the sensitivity in the identification of certain microorganisms. As N. Mancini et al. reviewed recently (4), several technologies are commercially available for the detection and identification of microorganisms related to bloodstream infections directly from whole blood. Among them, the most evaluated one in the hospital setting has been SeptiFast (Roche, Mannheim, Germany), leading to heterogeneous results (5). The low concentration of bacteria in blood of septic patients challenges even highly sensitive methods.

Recently, a new and promising technology has been developed that uses a broad-spectrum PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS; Ibis Biosciences, Abbott, Carlsbad, CA, USA) to detect and identify several hundreds of bacteria and Candida species in a clinical specimen with a single test (6-8). Using mass spectrometry, the mass of each PCR amplicon is determined and the nucleotide base composition is unambiguously calculated and compared to a database, achieving the identification.

We have designed the first observational study to assess, in the clinical setting, the usefulness of the PCR/ESI-MS technology in the diagnosis of bloodstream infections directly from the patient’s blood (N=247). The key findings in this study were: 1) the microbiological diagnosis of bloodstream infections could be anticipated in a significant proportion of cases in our setting using this technology in blood specimens (43.8% sensitivity and 88.4% specificity when compared to blood culture); and 2) the use of this technology could increase the diagnostic yield of conventional methods, as it detected microorganisms with clinical significance that were not isolated by blood culture in 13 blood specimens (a new microbiological diagnosis was achieved in nine patients adding up to 50% sensitivity and 93.8% specificity). While all of this work was done with a relatively small aliquot of EDTA blood (1.25 mL), newer methods in development using larger volumes of blood (up to 5mL) have shown 4´ better analytical limits of detection and ~85% clinical sensitivity (manuscript in preparation). The anticipation of the microbiological diagnosis of sepsis directly from patient specimen could lead to a rapid administration of an effective and more specifically-targeted antimicrobial therapy, with consequent reductions in the length of hospitalization and antibiotic treatment-related costs. Thus, the use of this technology in combination with traditional methods could have a positive impact on patient management and outcome as well as in public health costs.

Additionally, the described technology, being based on broad-range PCR, has the capability of not only detecting any pathogen but also quantifying them relatively against an internal calibrant in clinical specimens of different nature. Thus, this technology offers a high versatility for the diagnosis of infectious diseases as well as for the detection of previously uncharacterized pathogens. In fact, several assays have been designed for the broad identification of pathogens, targeted identification of groups of microorganisms (i.e.: respiratory viruses, food borne bacteria, etc.) and characterization of drug resistance/virulence (i.e.: flu, multi-drug resistant Mycobacterium tuberculosis, etc.).

Elisa Martró-png

Figure 1. Incidence of severe sepsis in U.S. in 2007 comparing with other major diseases.

Figure legend:

*www.cdc.gov, 2007 statistics  §Lagu T, et al. Crit. Care. Med. 2012;40;754-6

 

REFERENCES

1. Adhikari NK, Fowler RA, Bhagwanjee S, Rubenfeld GD 2010 Critical care and the global burden of critical illness in adults Lancet 376:1339-46. .

2. Kumar A, Ellis P, Arabi Y, Roberts D, Light B, Parrillo JE, Dodek P, Wood G, Kumar A, Simon D, Peters C, Ahsan M, Chateau D 2009 Cooperative Antimicrobial Therapy of Septic Shock Database Research Group. Initiation of inappropriate antimicrobial therapy results in a fivefold reduction of survival in human septic shock Chest 136:1237-48.

3. Kollef MH 1999 Antimicrobial therapy of ventilator-associated pneumonia how to select an appropriate drug regimen Chest 115:8-11.

4  Mancini N, Carletti S, Ghidoli N, Cichero P, Burioni R, Clementi M 2010  The era of molecular and other non-culture-based methods in diagnosis of sepsis Clin Microbiol Rev 23:235-51.

5. Chang SS, Hsieh WH, Liu TS, Lee SH, Wang CH, Chou HC, Yeo YH, Tseng CP, Lee CC 2013 Multiplex PCR system for rapid detection of pathogens in patients with presumed sepsis – a systemic review and meta-analysis PLoS One 8:e62323.

6. Ecker DJ, Sampath R, Massire C, Blyn LB, Hall TA, Eshoo MW, Hofstadler SA 2008 Ibis T5000: A universal biosensor approach for microbiology Nat Rev Microbiol 6:553-8.

7. Ecker DJ, Massire C, Blyn LB, Hofstadler SA, Hannis JC, Eshoo MW, Hall TA, Sampath R 2009 Molecular genotyping of microbes by multilocus PCR and mass spectrometry: A new tool for hospital infection control and public health surveillance Methods Mol Biol 551:71-87.

8. Ecker DJ, Sampath R, Li H, Massire C, Matthews HE, Toleno D, Hall TA, Blyn LB, Eshoo MW, Ranken R, Hofstadler SA, Tang YW 2010 New technology for rapid molecular diagnosis of bloodstream infections Expert Rev Mol Diagn  10:399-415.

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann