Recently there has been a rapid increase in technology for innovative molecular tests, most significantly associated with the use of nucleic acid amplification tests (NAATs), such as polymerase chain reaction (PCR). These new tests are becoming available with marked expansion of diagnostic capability for infectious diseases. Newer tests that may allow more rapid etiologic diagnosis include the newer generation of immunochromatographic urinary antigen tests as well as NAATs.
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A recent meta-analysis by Shimada and colleagues 21 summarized the performance characteristics of the Legionella urinary antigen as having very good specificity but lower sensitivity for L pneumophila serogroup 1; thus, it is better for ruling in than ruling out disease. A positive urinary antigen test result, in the appropriate clinical setting, virtually rules in legionellosis, but a negative urinary antigen test result does not rule out the presence of disease, as 26% of patients with confirmed legionellosis have a negative urinary antigen test result. One potential &#;unintended&#; adverse consequence of the availability of the Legionella urinary antigen test is decreased use of Legionella culture. All too often, clinicians order a urine antigen test without submitting or requesting a sputum culture. Both the urine antigen test and the Legionella culture should be performed for maximal effectiveness, especially if non&#;serogroup 1 L pneumophila is a consideration.
Presently the urinary antigen is the most used test in North America for detection of Legionella. 8 Although the test can reliably detect only one species, Legionella pneumophila, and only one serogroup, serogroup 1, it has significant advantages over previous &#;standard&#; tests (direct fluorescent antibody testing, serology, and culture), including its relatively low cost and rapid performance. Direct fluorescent antibody stains require substantial expertise for interpretation, and selection of reagents is critical. Culture on selective media detects all but very rare strains but is technically more demanding and requires 3 to 7 days. 16 Accurate interpretation of serologic tests requires comparison of acute and convalescent specimens, which is not relevant for clinical management. The Legionella urinary antigen test is 70% sensitive and greater than 90% specific for infections caused by L pneumophila serogroup 1 and should particularly be useful in the United States and Europe, as approximately 85% of community-acquired isolates are serogroup 1. 17 , 18 It may be less sensitive for nosocomial cases because of frequent involvement of serogroups other than serogroup 1. Urine is usually positive for antigen on day 1 of illness and continues to be positive for weeks. 19 , 20
The disadvantages of urinary antigen testing include the cost and the lack of an organism isolate for in vitro susceptibility tests. Notably, immunochromatography is not suitable for evaluation of therapeutic effect, because positive test results are obtained for several weeks to months after recovery. Moreover, the immunochromatographic assays are nonspecific for pneumococcal infections in children, particularly the very young, as nasopharyngeal carriage of S pneumoniae can cause false-positive results. 12 In one study, the presence of azotemia was an independent factor associated with a higher rate of a positive test for patients with bacteremia. 13 The investigators suggested this may have been because of increased concentration of urine for these patients, as most of the patients had reversible impaired renal function most likely caused by dehydration. Supporting the theory of the effect of concentrated urine, Gutierrez and colleagues 11 reported increased test sensitivity after urine concentration by centrifugation, and a study conducted by the manufacturer found that the test&#;s ability to detect pneumococcal antigen decreased with serial dilution. 15 Thus, patients may be more likely to test positive after urine sample concentration or before intravenous fluid resuscitation. Of note, pneumococcal vaccine may cause false-positive results in urine in the Binax NOW Streptococcus pneumoniae test in the 48 hours following vaccination. 15
The ICT urinary antigen test is particularly attractive for detecting pneumococcal pneumonia when cultures cannot be obtained in a timely fashion or when antibiotic therapy has already been initiated. In serial specimens from known bacteremic cases, the pneumococcal urinary antigen detected by ICT assay was still positive in 83% of cases after 3 days of therapy. 10 This form of urinary antigen testing has the principal additional advantages of rapidity (about 15 minutes), simplicity, and reasonable specificity in adults. Studies in adults have shown a sensitivity of 50% to 80% and specificity exceeding 90%. 11 , 12 , 13 In one study, the use of the ICT pneumococcal urinary antigen test increased the yield of etiologic diagnosis of patients admitted for CAP from 39.1% to 53.1%. 11 Of 269 patients in this study who had no defined etiology using conventional methods, 69 (25.7%) had a positive pneumococcal urinary antigen test. The immunochromatography assay is also highly accurate in diagnosing pneumococcal meningitis (95% sensitivity with cerebrospinal fluid, 57% sensitivity with urine, and 100% specificity). 14
Immunochromatographic (ICT) tests that detect soluble pneumococcal antigen or Legionella antigen in urine have been an important advance in the diagnostic assessments of these 2 pathogens. These tests are much less influenced by prior antibiotic therapy than sputum or blood culture. The ease of performing the ICT card-type urine test makes it ideal for use in emergency departments, long-term care facilities, and even physician offices (although presently they are not waived by the Food and Drug Administration [FDA] for nonlaboratory, office use).
Nucleic Acid Amplification Tests
The development of NAATs has been a major advance in the understanding of respiratory infections.8 PCR and related methodologies have revolutionized the field of molecular biology, and automated instrumentation has now been introduced successfully to the clinical laboratory setting. Molecular-based tests have moved from the research bench to the clinical diagnostic laboratory and now are becoming commercially available. Clinical application of these methods as comprehensive and rapid techniques may improve our ability to quickly and efficiently identify etiologic organisms associated with CAP. They may eventually have the potential to be point-of-care tests and allow pathogen-directed therapy at the time of initial administration of antimicrobial agents.
PCR directly detects microbial nucleic acid in clinical samples. The basic steps of PCR include DNA extraction from either a cultured pathogen or from a patient specimen sample and amplification of an established target gene.22, 23 Enzymes are used to copy this DNA via multiple rounds of replication, resulting in exponential amplification of the target sequence of interest. The PCR products can then be identified by gel electrophoresis and DNA sequencing.
Initially, PCR methods had several limitations, which included:
Requirement of adequate sample to detect DNA
Presence of PCR inhibitors in samples that can lead to false-negative results
Contamination, which can lead to false-positive results
Differentiation of colonization from true pathogens (eg, identification of S pneumoniae in a respiratory specimen; quantifying organisms may be helpful in this regard)
Equipment expense and requirement for trained personnel.
Lack of standardization of test methods (many hospital laboratories have their own methods that have not been validated in independent studies)
Only a few methods are presently approved by the FDA.
Many of these limitations have been addressed with advancements in methodology (see later in this article) or are expected to be resolved as technology improves. It is anticipated that molecular tests will be more available in the near future.
An important advance in NAAT technology has been the development of quantitative, real-time PCR.24 With this method, amplification and detection of the DNA sequence occurs in a single tube, thus simplifying the procedure, as gel electrophoresis sequencing is not needed. The reaction is performed with fluorescent-labeled DNA probes, which allow the number of gene copies to be determined. This increases the speed and efficiency of testing and reduces the risks of operator error and cross contamination. This process can be performed with faster turnaround times, allowing results to be used in a more prominent role in direct patient management. Another advancement has been the development of multiplex PCR systems, in which multiple DNA targets are assessed in one reaction without increasing the required amount of technician time.22, 23 Some commercially available assays can measure more than a dozen respiratory pathogens. These assays may also have the ability to recognize potential dual or triple infections in the same patient. Several commercial assays, which are based on automated extraction instruments, are available (but few are FDA approved at the time of this writing) and these vary according to methodology. Specifications for commercially available real-time PCR (including gene targets) are beyond the scope of this article, but readers are referred to other reviews for greater details.22, 23, 24 Presently FDA-approved tests are listed in
.
Table 1
BacteriaManufacturerTest NameMethodFrancisella tularensisIdaho Technology, IncSalt Lake City, UTJoint Biologic Agent Identification and Diagnostic System Tularemia Detection kitReal-time PCRMycobacterium tuberculosisGen-Probe, IncSan Diego, CAAMPLIFIED Mycobacterium tuberculosis Direct TestTranscription-mediated amplificationVirusAdenovirusGen-Probe, Inc (Prodesse)ProAdeno + AssayMultiplex Real-time RT-PCRAvian FluCenters for Disease Control and PreventionInfluenza A/H5Real-time RT-PCRInfluenza virus panelCenters for Disease Control and PreventionHuman Influenza virus Real-time RT-PCR Detection and Characterization PanelReal-time RT-PCRFocus Diagnostics, Cypress, CASimplexa Influenza testReal-time RT-PCRRespiratory virus panelLuminex Molecular Diagnostics, Toronto, CanadaxTAG Respiratory Viral Panel(Luminex LX 100/200) [includes Influenza A/H1, A/H3, A/ H1N1; Influenza B; Adenovirus; RSV A&B; Metapneumovirus, Parainfluenza 1,2,3; Rhinovirus]RT-PCRNanosphere, Inc Northbrook, ILVerigene Respiratory Virus Nucleic Acid test and Verigen Respiratory Virus TestMultiplex Gold Nanoparticle ProbesGen-Probe, Inc (Prodesse)ProFlu + Assay (Influenza A/B, RSV);
ProFast + Assay (Seasonal A/H1, A/H3, H1N1;
ProParaFlu + Assay (+Parainfluenza virus)Multiplex Real-time RT-PCROpen in a separate window
Data from FDA Office of In vitro Diagnostic Evaluation and Safety. Available at: www.fdagov/MedicalDevices/ProceduresandMedicalProcedures/DeviceApprovalsandClearance . Accessed December 16, .
Although PCR methods have been developed for several pneumonia pathogens, the clinical utility of these tests varies.25, 26, 27, 28, 29 There are several advantages of PCR testing methods as compared with standard microbiological culture methods in the detection of pneumonia pathogens (Box 1
). PCR is a potentially attractive diagnostic tool for rapid diagnosis because it does not rely on bacterial growth or the viability of the organism. Many pathogens, including Chlamydophila pneumoniae, Mycoplasma pneumoniae, and respiratory viruses, can be difficult to culture because of special growth requirements and slow growth. The time required for a final result is often too long to be clinically useful in the acute management of a patient. Real-time PCR has been shown to be as effective as culture methods for detecting these pathogens.24
Box 1
Advantages
Data from Chan YR, Morris A. Molecular diagnostic methods in pneumonia. Curr Opin Infect Dis ;20:157&#;64.
PCR for specific pathogens&#;bacteria
Streptococcus pneumoniae, the most common pathogen associated with CAP, is easily detected by PCR in respiratory specimens. PCR techniques based on amplification of the pneumolysin or autolysin genes are applicable for the diagnosis of pneumonia, otitis media, and meningitis. Autolysin and pneumolysin PCR using sputum have shown a high sensitivity (more than 80%) but a low specificity (30%&#;40%).30, 31 Interpretation of sputum PCR is limited by the difficulty in differentiating between pneumococcal colonization and true infection. On the other hand, in pleural fluid, PCR detects the pneumolysin gene with a sensitivity of 78% and a specificity of 93%.32 One approach that may help to resolve the problem of colonization versus infection is quantification of the target organism by real-time PCR. Yang and colleagues33 evaluated the utility of a real-time pneumolysin gene PCR test using sputum samples from patients admitted with CAP. Of 129 patients, 23% had S pneumoniae isolated from blood or sputum. The sensitivity and specificity using real-time quantitative PCR were 90% and 80%, respectively. Of note, PCR of blood samples from patients with S pneumoniae infection does not appear to be useful. One study compared the ICT S pneumoniae urine antigen test to PCR of blood in patients with bacteremic pneumococcal infections.34 The urinary antigen test was positive in 51 of 58 bacteremic pneumococcal cases (sensitivity, 88%; 95% confidence interval [CI], 77% to 95%), whereas PCR was positive in only 31 cases (sensitivity, 53.5%; 95% CI, 40% to 67%; P<.), and all of these had detectable urinary antigens. Both tests gave positive results in 2 of 51 control patients (referred to as other-organism septicemia), giving a specificity of 96% (95% CI, 86.5% to 99.5%). In 77 patients with nonbacteremic CAP, urinary antigen was detected significantly more often (in 21 patients [27%]) than a positive result by the PCR protocol (6 [8%]) (P<.002).34 A recent meta-analysis concluded that currently available PCR methods using blood samples for the diagnosis of invasive pneumococcal diseases lack the sensitivity and specificity necessary for clinical practice.35 To date there are no FDA-approved PCR tests for S pneumoniae.
There are several commercially available and/or institutionally developed NAATs for the atypical pathogens.26, 36, 37, 38, 39, 40, 41 However, none of these are officially FDA approved or available at the present time in the United States. Despite this, PCR is increasingly being recognized as a method of choice for detection of M pneumoniae and C pneumoniae. For both of these pathogens, diagnosis has usually relied on serology, which, as indicated previously, is usually not useful to the clinician during acute medical management. One large study examined the use of real-time PCR to detect M pneumoniae in children with CAP and found that PCR detected more cases than standard diagnostic techniques, which included culture.42 Of significance, PCR results were available within 2 hours&#;a major improvement over the 2 to 6 weeks usually required for serologic diagnosis. For C pneumoniae, culture on cell lines has traditionally been considered as a gold standard for diagnosis. However, cell cultivation is technically complex, and is associated with limited viability and slow growth such that it is restricted to specialized laboratories and is, therefore, not often used. For these reasons, PCR has become an option for diagnosis. There are numerous assays described (again non-FDA approved) but with various results of test performances and significant interlaboratory discordance of detection rates.26, 36, 37
For tuberculosis (TB), molecular techniques have been valuable.27 Because the organism can require 3 to 8 weeks to grow in culture, molecular techniques can be useful, allowing appropriate isolation, treatment, and disease control. FDA-approved PCR assays are available, which are most useful in patients with positive acid-fast smears. A positive PCR in a smear-positive patient is extremely likely to signal TB. Conversely, a negative PCR in a smear-positive patient likely signals infection with another species. Importantly, a negative PCR in a smear-negative patient does not rule out TB.
PCR for viruses
Perhaps the area where PCR can have the greatest impact on pathogen detection has been for respiratory viruses.43, 44, 45, 46, 47 The gold standard for viral identification has been conventional cell culture. However, even in specialized laboratories many viruses cannot be readily cultivated. Thus, many cases of viral illness go undetected and the exact incidence of viruses in CAP has remained uncertain. PCR offers the potential to significantly improve viral detection. For many respiratory viruses, PCR is now the most sensitive diagnostic approach. Most clinical microbiology laboratories use reverse transcriptase PCR (RT-PCR) assays to detect RNA viruses from clinical specimens.22 This technique is very sensitive and can detect transcript from a single cell. The method uses a reverse transcriptase enzyme to synthesize a complementary strand of DNA from an RNA template. The resulting complementary DNA is then used as the template in a PCR assay.
PCR was vital for epidemiology during the recent influenza H1N1 pandemic because commercially available rapid influenza detection tests (RIDTs) were found to be relatively insensitive (sensitivity ranging from 10%&#;70% depending in part on the method used).48 Several recent studies have demonstrated that when PCR methods are used for viral detection, there is a high frequency of viral identification from patients with lower respiratory tract infection. In a prospective study during a 12-month period (&#;) of adult patients admitted for CAP, etiology was assessed using molecular methods (PCR for viruses, Legionella, Mycoplasma, Mycobacterium tuberculosis, and S pneumoniae; urinary antigen assay for S pneumoniae and Legionella pneumophila, serogroup 1) in addition to conventional studies (blood, respiratory culture, serology) for 184 patients.49 A microbial etiology could be identified for 67% of all the patients. However, in 38 patients for whom all diagnostic methods were applied, a pathogen was identified for 89% of cases. The most frequently detected pathogens were S pneumoniae and respiratory viruses (
). Another study using NAATs for the identification of respiratory viruses in adult patients with CAP evaluated 183 adult patients with CAP, 450 control subjects, and 201 patients with nonpneumonic lower respiratory tract infection.47 At least one respiratory virus was identified in 58 patients with CAP (31.7%) compared with 32 (7.1%) in control subjects and 104 (51.7%) in patients with nonpneumonic lower respiratory tract infections (P<.01 and P<.01, respectively) (
). Of interest, the proportion of viruses identified in healthy subjects was not zero and this should be considered when interpreting corresponding proportions among patients.
Table 2
PathogenNo. (%)
N = 184Blood CultureRespiratory CultureUrinary AntigenPCRSerologyStreptococcus pneumoniae70 (38)&#;Mycoplasma pneumoniae15 (8)&#;&#;&#;87Haemophilus influenzae9 (5)&#;7&#;&#;&#;Moraxella catarrhalis7 (4)&#;7&#;&#;&#;Staphylococcus aureus4 (2)22&#;&#;&#;Legionella pneumophila3 (1)&#;12&#;&#;Streptococcus milleri group1 (0.5)1&#;&#;&#;&#;Nocardia sp1 (0.5)&#;1&#;&#;&#;Fusobacterium necrophorum1 (0.5)1&#;&#;&#;&#;Mycobacterium tuberculosis2 (1)&#;2&#;&#;&#;Viruses54 (29)&#;8&#;Influenza virus14 (8)&#;3&#;47Rhinovirus12 (7)&#;&#;&#;12&#;RSV7 (4)&#;1&#;15Parainfluenza virus7 (4)&#;1&#;15Coronavirus4 (2)&#;&#;&#;4&#;Metapneumovirus3 (2)&#;1&#;3&#;Adenovirus3 (2)&#;&#;&#;&#;3HSV 12 (1)&#;2&#;&#;&#;Enterovirus1 (0.5)&#;&#;&#;1&#;Open in a separate window
Data from Ref. 49
Table 3
VirusCAP (n = 183)Controls (n = 450)NPLRTI (n = 201)Coronavirus24 (13.1)17 (3.8)21 (10.4)RSV13 (7.1)4 (0.2)7 (3.5)Rhinovirus9 (4.9)9 (2.0)15 (7.5)Influenza A8 (4.4)2 (0.4)62 (30.8)Influenza B001 (0.5)Adenovirus3 (1.6)00Human metapneumovirus2 (1.1)00Parainfluenza virus (2 or 3)003 (1.5)TOTALViruses59 (32.2)32 (7.1)110 (54.7)Positive subjects58 (31.7)32 (7.1)104 (51.7)Open in a separate window
Data from Lieberman D, Shimoni A, Shemer-Avni Y, et al. Respiratory viruses in adults with community-acquired pneumonia. Chest ;138:811&#;6.
Thus, by supplementing traditional diagnostic methods with new PCR-based techniques, it is now apparent that viruses are becoming increasingly recognized as important causes of CAP in adults, but in standard practice, except for influenza virus, respiratory viruses are not often identified. However, as stated in a recent editorial commentary by Niederman, &#;This may change once these new diagnostic tools become more widely available, especially if they help us define an etiologic role of these pathogens and if they encourage the development of new and effective antiviral therapies.&#;50
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