The Role of the Laboratory in Prevention of Healthcare-Associated Infections
The Role of the Laboratory in Prevention of Healthcare-Associated Infections
Michael A. Pfaller
Daniel J. Diekema
The work required of the clinical microbiology laboratory and of the hospital infection prevention program (IPP) has become increasingly complex, demanding, and intertwined as we enter the second decade of the new century. To do their jobs effectively and efficiently, these two groups must work as a team, using the expertise from each discipline to improve patient care and safety. As always, the microbiology laboratory must be able to detect and identify microorganisms and determine their antimicrobial susceptibility profiles so that clinicians can diagnose and treat established infections and the IPP can monitor and prevent infections and exposures in healthcare settings.
The ever-increasing complexity and sophistication of modern medical care poses challenges for the IPP. The need for accurate and rapid diagnosis, identification, and antimicrobial resistance detection is more important than ever. New technology, developed to detect and characterize microorganisms, not only significantly improves the laboratory’s ability to keep up with the ever-changing array of healthcare-associated infection (HAI) pathogens, but also challenges laboratory and IPP personnel to use this technology in the most appropriate and cost-effective manner (1,2,3,4,5,6). In particular, molecular and proteomic technologies have enhanced the speed, accuracy, and sensitivity of detection methods and have allowed the laboratory to identify organisms that were previously unknown or “cryptic” as well as those that do not grow readily in culture (3,5,6). Molecular and proteomic techniques also enable the microbiologist to identify antimicrobial resistance genes and to perform strain typing of hospital organisms, thereby facilitating studies of HAI pathogen transmission (7,8,9).
In addition to performing their traditional roles in the clinical diagnostic laboratory, laboratory personnel must perform additional tasks that are critically important in supporting infection prevention activities: (a) participate in hospital-wide IPP activities, (b) recover and accurately identify responsible organisms to the extent that is needed in an outbreak investigation, (c) determine the antimicrobial susceptibility profile of HAI pathogens, (d) report in a timely fashion laboratory data relevant to infection surveillance and prevention, (e) provide additional studies, as needed, to establish the genetic relatedness of organisms, (f) provide, on occasion, microbiologic studies of the hospital environment and personnel, and (g) teach IPP personnel how to use laboratory resources appropriately during surveillance of HAIs and during epidemiologic investigations.
The clinical microbiologist (doctoral-level microbiologist, pathologist, microbiology supervisor, or designated laboratory personnel), hospital epidemiologist (or infectious disease clinician) and infection preventionist (IP) must work as a team to prevent and control HAIs effectively (10,11). Given continuous changes in HAI pathogens, antimicrobial resistance, medical care, and healthcare delivery, staff members from the laboratory and from the IPP must work to ensure collaboration and open communication. The relationship between the microbiology laboratory, the IPP, and increasingly the antibiotic stewardship program (12,13,14,15), is critical to the success of these important efforts to improve patient care, control costs, and preserve options for effective antimicrobial therapy. In this chapter, we discuss the microbiology laboratory’s role in this essential collaboration.
PARTICIPATION IN HOSPITAL-WIDE INFECTION PREVENTION ACTIVITIES
RELATIONSHIP OF THE LABORATORY TO THE INFECTION PREVENTION COMMITTEE
The clinical microbiologist (or microbiology supervisor in an institution that does not have a doctoral-level microbiologist) is an integral part of the IPP and thus must be an active member of the infection prevention committee. Because the infection prevention committee frequently bases its decisions on the results of microbiological tests, the clinical microbiologist must guide the committee in integrating culture results and selecting appropriate microbiologic approaches to solve specific problems. The microbiology laboratory can benefit if the IPP staff understand the routine processes in microbiology (e.g., timelines for the processing of blood, wound, or urine cultures and related techniques) (16). Specimen processing timelines enable IPP staff to set expectations of turnaround times for specific results and time constraints of microbiology test services, thus minimizing premature phone calls to the laboratory requesting culture information. Conversely, while serving on the committee, the microbiologist will learn about the problems confronting the IPP and thus will be better able to organize the laboratory’s response to such problems.
The microbiologist must educate the committee about several important issues. Because most IPP personnel have not worked in laboratories, the microbiologist should ensure that these individuals understand basic microbiology principles and techniques. The microbiologist must also explain the advantages and limitations, the scope and accuracy (i.e., sensitivity and specificity), and the costs of microbiologic methods used to detect, identify, and assess the antimicrobial susceptibility of HAI pathogens.
In addition, the microbiologist should inform the committee about changes in methods, reagents, or instrumentation that may substantially affect the laboratory’s ability to detect and characterize HAI pathogens. These include changes in the sensitivity and specificity of diagnostic methods, changes in antimicrobial susceptibility testing interpretive criteria, and taxonomic changes that may create confusion. An example of changes in testing and reporting criteria that directly affect infection prevention efforts is the recent change in the interpretive breakpoints for the Enterobacteriaceae and cephalosporins and carbapenems enacted by both the Clinical and Laboratory Standards Institute (CLSI) (17) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (18). The new (lower) breakpoints are intended to obviate the need for extended-spectrum β-lactamase (ESBL) confirmatory testing or modified Hodge testing (confirmation of carbapenemase) for clinical use. The result of this change in testing and interpretive criteria has been the loss of epidemiologic data for some IPPs that have come to rely on these confirmatory tests to guide prevention activities (19), and also has led to an increase in the number of isolates characterized as resistant to cephalosporins and carbapenems, and therefore potentially multidrug-resistant (MDR), with major implications for infection prevention (20,21,22,23). One institution recently reported that this change resulted in a 35% increase in the number of MDR-Gram-negative rods (GNR) identified and a concomitant increase in the hospitals’ use of contact precautions (20).
Members of the IPP must communicate with each other to accomplish their goals. Communication may be enhanced if the IPP staff regularly round in the laboratory to ask questions, review microbiologic and molecular testing results, and discuss current problems and views. Likewise, the microbiology staff should attend conferences at which IPP personnel discuss epidemiologic principles and contemporary topics. Unfortunately, several ongoing trends challenge these valuable personal interactions between the microbiology and IPP personnel (21). Consolidation of clinical microbiology laboratory services, off-site moves of microbiology laboratories, and total reliance on the electronic medical records to the exclusion of first-hand observation (e.g., review of culture plates or Gram stains) too often keep clinicians and infection prevention personnel out of the microbiology laboratory and keep microbiologists confined to the laboratory.
BUDGETARY CONSIDERATIONS
Given that most laboratories have limited financial and staff resources, the microbiologist must help the IPP staff and the committee understand the costs and appropriate indications for the microbiologic tests most commonly used to support epidemiologic investigations, so that these limited resources are used effectively. Costs for laboratory procedures that are not related directly to the care of patients (e.g., bacteriologic sampling of personnel or the environment) should be borne by a budget separate from the laboratory. To facilitate all of the microbiologic activities necessitated by an outbreak, the laboratory (or the hospital epidemiologist or the infection prevention committee, depending on the hospitals’ organizational structure) should have a contingency fund to enable personnel, materials, and space to be temporarily assigned to support the outbreak investigation (11,24). An investigation of an outbreak should not be financed by charging individual patients for cultures taken during the investigation. Viewing the clinical microbiology laboratory as an integral component of the IPP may help healthcare administrators understand the importance of adequate funding for the clinical microbiology laboratory, particularly as the laboratory’s activities increase to meet infection prevention priorities. Effective prevention saves not only lives but money, and these savings are rarely credited to the clinical microbiology laboratory (21).
ACCURATE IDENTIFICATION AND SUSCEPTIBILITY TESTING OF ORGANISMS INVOLVED IN HEALTHCARE-ASSOCIATED INFECTION
In many instances, the results of routine cultures are the first indication that patients have an HAI. For most epidemiologic investigations, the routine procedures performed in the microbiology laboratory are satisfactory. In selected instances, however, certain laboratory services and expertise that extend beyond routine practice and knowledge may be necessary. Regardless of which tests are performed, the laboratory must perform the tests quickly, accurately, and reproducibly to ensure that the IPP can identify and assess HAIs properly.
The spectrum of organisms causing HAIs is ever-changing and varies from region to region and even from hospital to hospital (Table 11.1). From the 1970s through 2000, the spectrum of HAI pathogens shifted from GNR to Gram-positive organisms and Candida emerged as a major problem (25,26). The incidence of HAIs caused by staphylococci and enterococci increased, as these organisms became progressively more resistant to antimicrobial agents, and IPP efforts focused on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) (27,28). Notably, infections due to Candida spp. steadily increased during this time (29) and were found to exceed those due to MRSA and VRE among intensive care unit (ICU) patients in the Extended Prevalence of Infection in Intensive Care (EPIC II) survey in 2007 (Table 11.1) (25,30). The improvement in methods to detect and identify viruses also highlighted these organisms as frequent HAI agents (31).
More recently, MDR-GNRs have become increasingly prevalent in many hospitals (13,14,32,33,34,35,36,37). These include ESBL- and carbapenemase-producing Enterobacteriaceae and multiple-or pan-drug-resistant nonfermenters such as P. aeruginosa, A. baumannii, and Stenotrophomonas maltophilia (Table 11.2) (13,33,36). The data from the EPIC II study show that the majority (62.2%) of infections in ICU patients were due to Gram-negative organisms in 2007 with 40% to 48% of all infections in Eastern European, Latin American, and Asian ICUs caused by P. aeruginosa and Acinetobacter spp. alone (Table 11.1).
The ESKAPE pathogens (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp.) are responsible for most HAIs in the modern hospital (Table 11.2) (36). The data from the EPIC II study indicate that the ESKAPE pathogens are involved in >70% of infections in ICUs (Table 11.1) (25). This group of organisms is at the heart of the global antimicrobial resistance problem, the members of which are often MDR by virtue of multiple acquired and transmissible resistance factors (Table 11.2).
Rapid and accurate identification of these key HAI pathogens, including their resistance mechanisms, is a key issue in both optimal patient care and in prevention efforts (7,38). Whereas semi-automated and automated identification and susceptibility testing systems are well-established in most clinical microbiology laboratories, recent improvements in some automated instruments have involved the expansion of their databases and the use of advanced technologies to decrease time to detection, greatly improving their clinical utility. Furthermore, a few commercial platforms have created novel decision support software that integrates identification and susceptibility test results with surveillance strategies for antimicrobial resistance and guidelines for therapy (39).
TABLE 11.1 Types of Bacteria and Fungi in Culture-Positive Infected ICU Patients According to Geographical Region: Epic II, 2007a,b
aData compiled from Vincent JL, Rello J, Marshall J, et al. International study of the prevalence and outcomes of infection in intensive care units. JAMA. 2009;302:2323-2329.
bAbbreviations: AFR, Africa; EEU, Eastern Europe; EPIC II, Extended Prevalence of Infection in Intensive Care; ESBL, extended-spectrum β-lactamases; ICU, intensive care unit; LAM, Latin America; MRSA, methicillin-resistant S. aureus; NAM, North America; OC, Oceania; VRE, vancomycin-resistant enterococci; VSE, vancomycin-susceptible enterococci; WEU, Western Europe.
cPercentages do not necessarily equal 100, because patients may have had more than 1 type of infection or microorganism.
In an effort to supplement the information supplied by Gram-stain of cultures and clinical material, novel culture-independent technologies have emerged to reduce the time required for identification of microorganisms, including peptide nucleic acid (PNA), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and rRNA probe matrices (40,41). Nucleic acid-based platforms now have been incorporated into the routine clinical laboratory to identify microorganisms by DNA target sequencing, to detect a pathogen by real-time amplification techniques, or to simultaneously detect multiple pathogens by arrays (2) (Tables 11.3 and 11.4). Nucleic acid sequence analysis of bacterial and fungal rRNA genes has expanded our understanding of the phylogenetic relationships among these organisms and is the new standard for bacterial and fungal identification (3). Another excellent technique for microorganism identification relies on the protein composition of a bacterial or fungal cell using matrix-assisted laser desorption ionization-timeof-flight (MALDI-TOF) mass spectrometry (MS) (1,6).
COLLECTION AND TRANSPORT OF SPECIMENS
Specimen collection, transport, and handling must be of sufficient high quality to provide valid data (42,43,44). Specimens that are not collected or transported properly may give inaccurate results, even when handled as well as possible once they reach the laboratory. In turn, these inaccurate results may lead to improper clinical decisions by physicians, unnecessary labor by laboratory and IPP personnel, and unnecessary patient charges. Many tests may have very specific requirements for specimen type, collection, and transport to the laboratory. This is especially true for most molecular tests (45).
Many HAI pathogens (e.g., coagulase-negative staphylococci, Candida) also commonly colonize patients’ skin or mucous membranes and can easily contaminate cultures if specimens are not collected or handled properly. If contaminants are considered to be infecting organisms and result in a patient meeting the HAI definition, HAI rates may be inflated through misclassification (16,46).
TABLE 11.2 Antimicrobial Resistance Problems Associated with the ESKAPE Pathogensa
Pathogen
Antimicrobial Agent
Resistance Mechanisms
Enterococcus faecium
Ampicillin
Aminoglycosides
Vancomycin
Mutation in Pbp5
Enzymatic modification
Altered peptidoglycan cross-link target
Staphylococcus aureus
Aminoglycosides
Fluoroquinolones
Linezolid
Penicillin
Oxacillin
Sulfonamides
Tetracyclines
Trimethoprim
Vancomycin
Enzymatic modification
Mutation in gyrA leading to reduced binding to active site; efflux.
Mutation in rRNA leading to reduced binding to active site.
β-lactamase
Altered PBP (PBP2a)
Mutation or recombination of genes encoding DHPS
Efflux
Mutations in gene encoding DHFR
Altered peptidoglycan cross-link target
Klebsiella pneumoniae
β-lactams
Amp C enzymes
ESBL
Altered/decreased porins
Efflux
Carbapenems
Metallo-β-lactamases
KPC-type β-lactamases
Altered/decreased porins
Fluoroquinolones
Altered target, protection from DNA binding, efflux
Acinetobacter baumannii
Aminoglycosides
β-lactams
Carbapenems
Enzymatic modification
Numerous β-lactamases
Metallo-β-lactamases
OXA carbapenemases
Porin alteration
Fluoroquinolones
Altered target
Pseudomonas aeruginosa
Aminoglycosides
Changes in outer membrane permeability, efflux, enzymatic modification
The laboratory must monitor specimen quality carefully and also work closely with inpatient and ambulatory care units to develop and enforce strict criteria for acceptable collection and handling of clinical specimens. This is necessary to ensure that the laboratory information presented to the clinician and the IPP reflects organisms that are actually associated with the patients’ site of culture rather than contaminants.
Certain laboratory findings suggest specific handling errors (47). For example, a persistent failure to isolate organisms from patient specimens with pathogens seen on Gram-stain suggests inadequate transport media, delay or inappropriate refrigeration of specimens in transit, errors in staining, contaminated reagents, or inadequate culture techniques. Likewise, the frequent recovery of ≥3 different organisms in clean-voided, midstream urine specimens suggests unsatisfactory methods of specimen collection, delay in transporting specimens to the laboratory, or delay in culturing them.
Specimen collection and handling should be assessed regularly to detect and correct such problems; the frequency with which probable contaminants are isolated from clinical specimens can be a measure of the quality of specimen collection in a specific hospital area. Addressing these issues is another area where laboratory and IPP personnel can work together to improve results. Bringing such data to a multidisciplinary level by involving IPP personnel has the potential to decrease contamination rates (17). For example, determining the frequency of urine specimens with characteristics suggestive of contamination can identify patient care units that may require further evaluation and, if necessary, in-service education programs conducted by laboratory or IPP personnel.
Many hospitals also monitor specimen transport time and use this information to avoid culturing of old, inadequate specimens. Evaluation of test turnaround time has become an important element of laboratory quality assurance (48).
TABLE 11.3 FDA-Cleared or-Approved Molecular Diagnostic Tests for Infectious Diseasesa,b
Organisms Detected in Clinical Material or Identified from Culture
Test Method
Detected
Identified
bDNA
HCV
HIV
HC
Chlamydia trachomatis
Neisseria gonorrhoeae
CMV
HPV
HDA
HSV 1 and 2
HPA
C. trachomatis
Mycobacteria spp. (6 species)
N. gonorrhoeae
Blastomyces dermatitidis
Group A Streptococci
Coccidioides immitis/posadasii
Group B Streptococci
Cryptococcus neoformans
West Nile virus (blood donation)
Culture confirmation of 9 different bacteria
Invader® Chemistry
HPV
Loop amplification technology
Clostridium difficile
Group B Streptococci
NASBA
MRSA screen
CMV
Enterovirus
PCR
Bacillus anthracis
MRSA
Bordetella pertussis
Culture confirmation of >1,000 different species
Chlamydophila pneumoniae
C. difficile
Coxiella burnetii
C. trachomatis
Francisella tularensis
Group B Streptococci
Leishmania
MRSA
M. tuberculosis
Enterococcus vanA
Adenovirus
Avian Flu
Enterovirus
HBV
HCV
HIV
HBV/HCV/HIV (for blood and tissue donation)
HSV 1 and 2
Human Metapneumovirus
HPV
Influenza Virus Panel
Influenza Virus-H1N1
Respiratory Virus Panel
West Nile Virus (blood donations)
PNA-FISH
M. tuberculosis
Candida spp. (5 species from blood cultures)
Enterococcus faecalis
Escherichia coli
Klebsiella pneumoniae
Pseudomonas aeruginosa
Group B Streptococci
Staphylococcus aureus
Other Enterococci
Other Staphylococci
SDA
C. trachomatis
N. gonorrhoeae
HSV 1 and 2
TC
C. trachomatis
N. gonorrhoeae
HBV/HCV/HIV (blood and tissue donations)
HPV
West Nile Virus (blood donations)
TMA
C. trachomatis
N. gonorrhoeae
Mycobacterium tuberculosis
Trichomonas vaginalis
HCV
HPV
West Nile Virus (blood donations)
Multiplex gold nanoparticle probes
Influenza Virus Panel
Respiratory Virus Panel (7 viruses)
West Nile Virus (blood donations)
MRSA
Culture confirmation of 9 different species of Gram-positive cocci, mecA, vanA, vanB
aModified from http://www.amp.org accessed 1 July 2012
bAbbreviations: bDNA, branched chain DNA Signal Amplification; CMV, cytomegalovirus; HBV, hepatitis B virus; HC, hybrid capture; HCV, hepatitis C Virus; HDA, Helicase-Dependent Assay; HIV, human immunodeficiency virus; HPA, Hybridization Protection Assay; HPV, human papillomavirus; HSV, herpes simplex virus; MRSA, methicillin-resistant S. aureus; NASBA, Nucleic Acid Sequence-Based Amplification; PCR, polymerase chain reaction (includes real-time PCR, multiplex PCR, and RT-PCR); PNA-FISH, peptide nucleic acid-fluorescent in situ hybridization; SDA, Strand Displacement Amplification; TC, Target Capture; TMA, Transcription Mediated Amplification.
INITIAL EVALUATION OF SPECIMENS
Assessing the quality of specimens at the time that they are received in the laboratory is one of the best ways to evaluate their suitability for further microbiologic work up. Microscopic review of Gram-stain of sputum specimens is a proven means for determining the adequacy of these specimens (42,49); specimens identified as inadequate (multiple squamous epithelial cells, no neutrophils) are not processed further and do not confuse either clinician or epidemiologist. Scoring systems for use in determining acceptable wound, vaginal, cervical, or other specimens have also been described (42,50). Application of such criteria ensures that the information generated from the specimens that are processed completely will more likely correlate with true infecting organisms and will reduce unnecessary laboratory costs. Repeat specimen collection should be requested for inadequate specimens, and further processing of organisms isolated from poor specimens (e.g., species identification, susceptibility testing) should be delayed or eliminated. The culture report should alert the clinician regarding the questionable value of the specimen so that results will be used cautiously, if at all, for guidance in diagnosis and therapy. Efforts such as these substantially improve the quality of the microbiologic results provided and reduce errors in the diagnosis and use of unnecessary antimicrobial therapy.
RAPID TESTS FOR ORGANISM DETECTION AND IDENTIFICATION
Arguably, the most rapid and cost-effective test performed in the clinical microbiology laboratory is direct microscopic examination of clinical material. Unfortunately, microscopy is inherently insensitive and nonspecific and so a number of different rapid culture-independent methods have been developed that employ immunologic, molecular, or proteomic methods in order to enhance the laboratory’s ability to diagnose and identify HAI pathogens (1,2,3,4,5,6,51,52,53,54,55).
Although no formal definition of “rapid” exists for describing the time required for results to be generated, most clinicians and microbiologists consider rapid results to be available within 2 to 4 hours (4,39). Presently, immunologic methods for rapid diagnosis directly from clinical material are used to detect a variety of viruses, S. pneumoniae, N. meningitidis, Group B streptococcus, L. pneumophila, C. difficile toxin, Aspergillus, Candida, C. neoformans, P. jirovecii, Giardia, Cryptosporidium, and E. histolytica, to name a few (51).
Although long considered to be of great potential for enhancing the ability of the microbiology laboratory to rapidly detect and identify infectious agents (4), the use of molecular methods in infectious disease diagnosis has increased substantially only in the past 5 to 10 years. The growth in the number of commercially available test kits and analytespecific reagents (ASRs) has facilitated the use of this technology in the clinical laboratory (Tables 11.3 and 11.4) (56). Technological advances in real-time PCR techniques, automation, nucleic acid sequencing, multiplex analysis, and MS have pushed the field forward and created new opportunities for growth.
Although the literature is replete with reports of “laboratory designed” or “homebrew” methods that are directly applicable to the needs of infection prevention (2,56), most commercial molecular diagnostic tests are of limited or no use for infection prevention purposes (Tables 11.3 and 11.4). Among the FDA-cleared or approved tests listed in Table 11.3, the most useful for infection prevention purposes include rapid identification of ESKAPE pathogens by PNA-FISH and molecular detection of MRSA, M. tuberculosis, C. difficile, enterovirus, influenza, and other respiratory viruses.
TABLE 11.4 Commercial Molecular Assays for Diagnosis of Sepsisa,b
Assay
Manufacturer
Method
Detectable Pathogens
Detection Limit (cfu/mL)
Turnaround Time (hr)
PERFORMED ON POSITIVE BLOOD CULTURE BOTTLES
BD Gene Ohm Staph SR
BD Diagnostics, Sparks, MD
Real-time PCR
S. aureus, MRSA, MSSA
NA
3
Xpert MRSA/SA BC
Cepheid, Sunnyvale, CA
Real-time PCR
S. aureus, MRSA, MSSA
NA
3
PNA-FISH
AdvanDX, Woburn, MA
Fluorescence-based hybridization with PNA probes
Identification of 7 bacteria and 5 Candida species
NA
3
Hyplex BloodScreen
BAG, Lich, Germany
Multiplex PCR with hybridization on an ELISA plate
10 different pathogens and mecA gene
NA
3
Prove-it Sepsis
Mobidiag, Helinski, Finland
Multiplex PCR with hybridization on a microarray
50 different pathogens and mecA
NA
3
Verigene Gram-Positive Blood Culture Test
Nanosphere, Inc. Northbrook, IL
Multiplex gold nanoparticle probes
11 different pathogens and mecA, vanA, vanB genes
NA
3
PERFORMED DIRECTLY ON WHOLE BLOOD
SepsiTest
Molzym, Breman, Germany
Broad-range PCR with sequencing
>300 different pathogens
20-40 for S. aureus
8-12
Vyoo
SIRS-Lab, Jena, Germany
Multiplex PCR with gel electrophoresis
>40 different pathogens and mecA, vanA, vanB, vanC and blaSHV genes
3-10
8
LightCycler SeptiFast Test
Roche Molecular Systems, Branchburg, NJ
Multiplex real-time PCR
25 different pathogens
3-30
6
aAdapted in part from Mancini N, Carletti S, Ghidoli N, et al. The era of molecular and other non-culture-based methods in diagnosis of sepsis. Clin Microbiol Rev. 2010;23:235-251.
bAbbreviations: ELISA, enzyme linked immunosorbent assay; MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; NA, not available; PCR, polymerase chain reaction; PNA-FISH, peptide nucleic acid-fluorescent in situ hybridization.
PNA-FISH probes have been used for identification of S. aureus, E. coli, P. aeruginosa, and C. albicans directly from positive blood culture bottles (Table 11.4) (57,58,59) and direct detection of M. tuberculosis in smear-positive sputum specimens (60). PNA-FISH probes for rapid (˜3 hours), direct identification of S. aureus, coagulase-negative staphylococci, E. faecalis, E. coli, K. pneumoniae, P. aeruginosa, C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei from positive blood culture bottles and S. agalactiae from Lim broth cultures are available from AdvanDx (Woburn, MA) (Table 11.3) (59,61,62). Whereas the use of PNA-FISH probes can provide identification within 3 hours, the requirement for prior amplification in culture lessens the impact of this technology.
PCR coupled with nucleic acid sequencing has proven to be an excellent means of rapidly identifying bacteria and fungi from blood culture or other cultural materials, and this approach can be considered the new standard for bacterial and fungal identification (56). Applied Biosystems (Foster City, CA) has developed ribosomal gene sequencing kits for bacteria and fungi. A sequence from an unknown organism is compared with either a full or partial 16S rRNA (D2 large-subunit rRNA for fungi) sequence from over 1,000 type strains by using the MicroSeq analysis software (63). The software analysis provides percent base pair differences between the unknown agent and the 20 most closely related organisms, alignment tools to show differences between the related sequences, and phylogenetic tree tools to verify that the unknown organism clusters with the 20 closest organisms in the database. Continual refinements in methods and software, and decreases in cost, should lead to more widespread use of sequence-based approaches to microbial identification.
As seen in Table 11.4, two additional kits that use PCR and sequence-based identification of organisms from positive blood cultures are commercially available in Europe: the Hyplex BloodScreen (BAG, Lich, Germany) and the Prove-it Sepsis kit (Mobidiag, Helsinki, Finland). Both methods use multiplex PCR with subsequent hybridization to identify between 10 and 50 pathogens plus the mecA gene. The turnaround time for both assays is 3 hours and they both claim sensitivities of 94% to 100% and specificities of 92% to 100% (54). The Hyplex assay also is available in formats to allow the detection of other resistance markers, such as van genes and several β-lactamase genes. The Verigene Gram-Positive Blood Culture Test has recently been cleared by the FDA for the identification of 11 different Gram-positive bacteria and 3 resistance genes from positive blood cultures. This assay uses PCR plus nanotechnology to provide rapid, accurate results. Again, the clinical usefulness of these assays is limited by their use only for positive blood cultures and not directly for blood samples.
The most recent approach to the rapid identification of bacteria and fungi from culture is the use of proteomics facilitated by MALDI-TOF MS (1,6). MALDI-TOF systems use mass measurements of nucleic acids or proteins of bacteria or fungi and have emerged as robust, rapid and inexpensive methods to detect and characterize a wide range of organisms including anaerobes, nonfermenting GNR, staphylococci, Candida, and Aspergillus spp. (64). The use of MALDI-TOF MS to identify bacteria has been shown to be highly accurate relative to 16S rRNA sequencing and capable of delivering an identification of a pre-cultured organism in 6 minutes at a cost that is one-quarter of that of conventional identification (1). Some methods for the processing of positive blood culture samples have been published, but direct testing of other samples, such as urine, will require additional development (6,64,65). Novel applications for this technology are being developed and include microbial strain typing, antimicrobial susceptibility testing, and the study of virulence profiles (6).
Rapid methods for detecting important antimicrobial resistances have been developed, with most of the current focus being on rapid detection of MRSA (38,66,67) and VRE (5,38). A positive result from any of these tests allows clinicians to implement appropriate isolation precautions quickly in order to prevent the spread of resistant organisms. Whereas PCR-based methods have been developed that allow rapid characterization of common and emerging resistance mechanisms, including a variety of β-lactamases and carbapenemases in key HAI pathogens (Table 11.4), for the most part, they are not yet readily available outside of the research or reference laboratory setting (8).
Whereas blood cultures remain the current gold standard for the diagnosis of bloodstream infection (BSI), the requirement for bacterial or fungal growth to optimize detection and identification makes this key diagnostic test of little use in the rapid diagnosis of infection (54). An ideal test for both diagnostic and infection prevention purposes would be one that could be applied directly to a sample of the patients’ blood (or other clinical material) and that would detect the presence and identity of the etiologic agent within 2 to 4 hours or at least on the same day that the specimen is processed (3,5,54). Many such assays using nucleic acid-based technologies have been developed in the research setting (54,63,68,69); however, aside from application in virology (52,56
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