CURRENT DIAGNOSTIC TOOLS: LIMITATIONS AND PROBLEMS
With the exception of urinary antigen testing our current set of diagnostic tools have been available for nearly a century, although there have been serial improvements in their performance over that time. All have been extensively evaluated as far as their performance and impact on clinical care.
Sputum Gram stains
Sputum Gram stain can be highly suggestive of pneumococcal pneumonia if the sample is of high quality and predominantly shows Gram-positive diplococci. A meta-analysis study by Reed et al. performed between 1966 and 1993 showed varied sensitivity (15–100%) and specificity (11–100%) for sputum Gram stains. Additional prospective studies since with high-quality sputum specimens have shown comparatively higher sensitivity (57–82%) and specificity (93–97%); however, these may well represent high estimates due to publication bias [8–10]. Although it has its strong advocates, direct microscopic examination of Gram-stained specimens has little place in usual clinical practice. Difficulty in collection of adequate quality sputum and treatment with antimicrobial therapy prior obtaining sputum specimens may produce low diagnostic yields. In an outpatient setting, the availability of 24-h laboratory facilities and experienced microbiologists are significant limitations. Moreover, it may be difficult to collect high-quality sputum from children, and even in adults getting quality sputum from sick patients to the laboratory in a timely manner is often difficult [11▪]. Although a Gram stain may indicate the presence of S. pneumoniae, it gives no data as to antibiotic sensitivity, limiting the ability to narrow antibiotic coverage from a third-generation cephalosporin or β-lactam. Finally, a large amount of data over the past decade suggest that β-lactam monotherapy for pneumococcal pneumonia is associated with worse clinical outcomes [1▪], recently confirmed by a randomized, placebo-controlled trial [12▪▪]. Therefore, even if S. pneumoniae is suspected based on a Gram stain, combination antibiotic therapy with a β-lactam and a macrolide is the preferred option in hospitalized patients [1▪]. Although sputum Gram stain does not have a role in routine management of patients with CAP for all the reasons discussed above, in the critically ill patient it is still recommended given the potential that it might suggest an otherwise unsuspected Gram-negative pathogen such as Pseudomonas. For this reason sputum (or endotracheal aspirate) Gram stain and culture are obligatory in the ICU setting.
The main limitation of all culture techniques is the time delay in obtaining results such that using conventional techniques they have no prospect of altering empiric therapy. Not surprisingly, given the difficulties in obtaining specimens, reduction in yield with prior antibiotics and the same caveats with respect to copathogens and the data on combination antibiotic therapy, there is no evidence other than anecdotal case reports that sputum cultures alter clinical practice. Attempts to speed up the assessment of cultures with new platforms will be discussed later.
Despite being a gold standard, the lack of impact of blood cultures on patient management offers salient lessons in developing new tests. Up to 70% of positive blood cultures in some studies are false positives, usually due to coagulase negative staphylococci . With increased awareness of multi-resistant Staphylococcus aureus (MRSA) in the setting of HCAP, initial detection of a possible Staphylococcus in a blood culture is now much more likely to lead to overtreatment with vancomycin or one of the newer anti-Staphylococcal antibiotics than in the past, making false-positive blood cultures a much greater clinical problem.
The primary limitation of blood cultures is the time delay such that results do not impact on empiric therapy. Despite the agreed high specificity of blood cultures when they detect S. pneumoniae (or another known pathogen), clinicians largely ignore the results and rarely narrow antibiotic therapy or otherwise change management after empiric therapy has been initiated [14–16]. Another limitation of blood cultures is their low positive rate, estimated at less than 10% for CAP due to S. pneumoniae[11▪], and overall typically significantly less than 5% of cases in large etiological studies. Like sputum cultures, prior antibiotic exposure greatly decreases the positive rate, which presents a major problem in up to half of patients in most series, and the caveats of copathogens and evidence for combination antibiotic therapy further limit the potential for blood cultures to impact on empiric therapy decisions, with the rare exception of unusual pathogens resistant to standard empiric regimens. Although the taking of blood cultures has been identified as a predictor of better outcomes in several studies, given the paucity of evidence that they change patient management , it is more likely that this represents one component of a bundle of behaviors and actions associated with overall better medical care rather than a specific cause and effect relationship .
BinaxNOW Streptococcus pneumoniae
BinaxNOW (Binax Inc., Portland, Maine, USA) S. pneumoniae is an immunochromatographic test for the presence of S. pneumoniae C polysaccharide antigen. Applied to an initial urine sample, it provides rapid potential detection of S. pneumoniae. Pooled analysis of BinaxNOW S. pneumoniae indicates it has a sensitivity of 75–85% in bacteremic patients and 50–80% in nonbacteremic patients. The sensitivity decreases further after antibiotic use. Overall the reported specificity, positive predictive value and negative predictive value of the BinaxNOW S. pneumoniae range between 92–95, 70–80 and 90–96%, respectively [19▪]. It is also important to note that children have much higher rates of false-positive urinary antigen tests associated with nasopharyngeal carriage [20–22], and prolonged urinary antigen excretion has been noted which may lead to false-positive results in patients with serial chest infections .
Despite the relative ease of use and quick turn around time, most studies have shown that the routine use of the urinary pneumococcal antigen testing in hospitalized patients with CAP has limited impact on antibiotic treatment. Piso et al. showed that pneumococcal urinary antigen testing did not lead to cost savings or narrowing of antibiotic prescriptions. A report by Sinclair et al.[24▪] also demonstrated that there is currently no evidence that the introduction of BinaxNOW S. pneumoniae influenced physicians’ prescribing habits. Although there are some reports of physicians narrowing antibiotic therapy based on a positive urinary antigen result , the recent data on combination antibiotic therapy for pneumococcal disease already discussed questions the appropriateness of this response. As detection of S. pneumoniae also does not give any data about antibiotic sensitivity, this further limits the potential to narrow therapy in regions with significant antibiotic resistance.
Overall the BinaxNOW S. pneumoniae is a useful etiological test, but has very limited ability to alter clinical management in the setting of CAP. It may have greater utility to stop the use of anti-MRSA and anti-gram-negative coverage in HCAP, but this has not been properly assessed.
Pfizer's 13-plex urine antigen diagnostic assay
Pfizer's 13-plex UAD assay is able to simultaneously detect 13 different S. pneumoniae serotype-specific capsular polysaccharides included in its 13-valent pneumococcal conjugate vaccine by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) in human urine. A proposed advance on the Binax urinary antigen assay of increased specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres .
Validation experiments suggest the Pfizer UAD assay may be superior to the Binax product with 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. The Pfizer UAD assay also identified S. pneumoniae (13 serotypes) in a percentage of patients with nonbacteremic CAP. Since the Pfizer assay only detects 13 serotypes, its performance against the Binax competitor will depend on the proportion of pneumococcal disease from this subset. However, despite possible improved overall sensitivity, the Pfizer UAD suffers from the same limitations as the Binax product with respect to clinical utility in altering antibiotic therapy.
NEW DIAGNOSTIC TECHNIQUES: WHAT REALLY IS ‘NEW’ OUT THERE AND RAPID TESTS IN EVOLUTION
A host of new assays using conventional and novel platforms that can detect potential pneumonia pathogens are in varying stages of development. The extent of data available varies considerably, but we will review what is published and what can be extrapolated from the available data.
Rapid influenza tests
Influenza remains a major cause of CAP, and particularly since the H1N1 09 pandemic there has been anecdotal evidence of a marked increase in the use of empiric antiinfluenza therapies, and especially oseltamivir, in hospitalized patients . A fast and reliable diagnostic test for influenza is therefore attractive not only to prescribe antivirals appropriately (for treatment and prophylaxis) but also to aid in the allocation of respiratory isolation beds that are often in limited supply, especially in influenza season.
In the United States, there are currently 14 approved rapid influenza tests primarily based on the detection of influenza antigens in respiratory secretions. Most available assays have been compared to a gold standard of real-time reverse transcription PCR in the same sample. The sensitivity varies between 10 and 75% depending on age, quality of sample and duration of symptoms. Complicating assessment of the utility of these assays is that the performance seems to vary between influenza strains, and unfortunately during the H1N1 09 pandemic they were less than optimal [28–31].
A recent meta-analysis of 159 published studies of rapid influenza tests found the pooled sensitivity, specificity, positive and negative predictive values to be 62, 98, 34 and 38%, respectively . Not surprisingly, there is little evidence that rapid influenza tests are currently used by clinicians to alter patient management [33▪▪]. It is important to note, however, that this is a rapidly changing field with advances in immunochoromatographic assays and now potential point-of-care nucleic acid amplification tests (NAATs, as discussed below). More recent publications suggest there are incremental improvements with a range of sensitivity from 68 to 79% and specificity of 99–100% [34▪,35]. This is clearly one area where there may be significant advances in the next few years.
Nucleic acid amplification tests
There are many NAATs in use for the detection of respiratory pathogens. These are a combination of ‘home grown’ and commercially available assays. Assays for almost all described pneumonia pathogens have been developed and published. Although not new, there continues to be significant development in the platforms for performing NAATs that promise faster turn around times and greater simplicity of use. The latter is particularly important given the heavy reliance on specialized microbiology laboratories to perform NAATs.
As NAATs do not require the organism to be viable, prior antibiotic therapy does not have a significant impact on their performance as it does for traditional culture-based techniques. In the setting of CAP the most well described assays are those for detecting S. pneumoniae and those detecting common respiratory viruses such as influenza.
NAATs for pneumococcus have focused on multiple genetic targets in blood and respiratory tract samples. The pneumolysin gene (ply), autolysin gene (lytA), pneumococcal surface adhesin A gene (psaA), wzg/cpsA and the Spn9802 gene fragment have all been used as PCR targets to detect S. pneumoniae. The best performing assays on whole blood typically have a detection rate approximately twice that of the blood culture rate and do not have a false-positive rate in healthy controls [36,37].
Another advantage of NAAT is the ability to quantify the amount of target present in the initial sample, giving a crude estimate of the amount of bacteria in the sample. Several studies have now demonstrated that the quantitative count of S. pneumoniae in blood [36–38] and sputum  is a strong predictor of clinical outcome in patients with pneumonia. The potential for the quantification of bacterial load to aid in the risk stratification of patients with pneumonia remains an active area of research interest but requires the same improvements in timeliness and ease of use as for existing NAATs.
Given the concern about the overuse of anti-MRSA coverage in the setting of HCAP, NAATs could be used to screen patients for possible MRSA infection from nasal swabs. There is evidence that in the setting of CAP a negative PCR for MRSA has a high negative predictive value allowing discontinuation of anti-Staphylococcal antibiotics [39▪]. Again as platforms improve in efficiency and price this will be a significant area of interest from both a pharmaco-economic and antibiotic stewardship perspective.
An additional appeal of NAATs is the ability to multiplex – the running of simultaneous assays on the one specimen that can detect a variety of pathogens including bacteria and viruses. Unfortunately, with the technology readily available to date there is a general decline in the performance of individual assays as an increasing number are combined such that the goal of a ‘single test to diagnose all pathogens’ remains elusive. With improved platforms there have been some advances with multiplexed assays and a handful are approved in the United States with the ability to detect more than three respiratory viruses, although each still has its problems with sensitivity and specificity depending on the viral target and the gold standard employed .
A variety of new platforms have been developed to speed up pathogen identification and even antibiotic sensitivity testing. In general, these can be categorized into those specifically designed to speed up pathogen recognition from positive blood cultures [e.g. The Verigene Gram Positive Blood Culture Nucleic Acid Test (Nanosphere Technology, USA), Prove-it Sepsis StripArray technology (Mobidiag, Finland) and FilmArray (BioFire Diagnostics, USA), and those designed for clinical samples such as sputum, blood or urine – e.g. GeneXpert (Cephenid, USA)], SeptiFast (Roche Diagnostics, Germany), SepsiTest (Molzym, Germany), Curetis Unyvero (Curetis AG, Holzgerlingen, Germany) and VYOO (SIRS Lab, Germany). Data in the specific setting of CAP are relatively limited for all of these platforms; however, there is a reasonable body of evidence in sepsis generally that is very likely to be applicable to pneumonia. A number of platforms are worth more detailed explanation.
GeneXpert is an ‘all in one’ platform designed as a point-of-care testing station. The clinical sample is placed in a cartridge, which plugs into the platform that runs the assay without the need for further processing or expert microbiological assistance. With a turn around time less than 2 h, results can be available fast enough to impact on empiric therapy. Extensively evaluated for the diagnosis of tuberculosis, including multidrug resistant tuberculosis, where it has been proven to be highly sensitive and specific , the range of pathogens that assays have been developed for the GeneXpert platform has been extended to include pathogens relevant to pneumonia. An influenza A and B GeneXpert assay was evaluated in comparison with two commercially available rapid antigen tests and found to have excellent sensitivity (97.3 and 100% for A and B, respectively) and specificity (100% for both) . Early results from the Xpert MRSA assay are also encouraging, although as yet not applied to the setting of CAP or HCAP [43▪,44].
FilmArray is another novel ‘all in one’ multiplex PCR platform with minimal technical expertise required and a turn around time of approximately 1 h. Manual handling is very limited as with GeneXpert and a variety of panels are available. The commercially available respiratory panel detects 20 viral and bacterial pathogens, but not S. pneumoniae, MRSA or gram negatives. The performance of the respiratory panel has been compared to ‘in house’ PCR tests with favorable results . Analysis of a FilmArray pneumonia panel that can detect 24 common respiratory pathogens including eight gram-positive bacteria, 11 gram-negative bacteria and five Candida species has also been published in abstract form in the setting of ventilator-associated pneumonia .
The Curetis Unyvero P50 pneumonia cartridge can detect 17 bacterial and fungal pathogens and 22 antibiotic resistance markers from respiratory samples in a single run in approximately 4 h [47▪]. The panel includes S. pneumoniae, Hemophilus influenzae, S. aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and atypical bacteria. It also includes a number of antibiotic resistance determinants. A recent study in the setting of nosocomial pneumonia found physicians changed antibiotic therapy based on the Unyvero test result in 33 (67.3%) of patients, which is substantially greater than seen in previous studies with other diagnostic methods [47▪]. A second study in the setting of severe hospitalized pneumonia [without categorization as CAP, HCAP, hospital acquired pneumonia (HAP) or ventilator associated pneumonia (VAP)] found compared to current standard of care methods the Unyvero system had a 70.6% sensitivity and 95.2% specificity .
Mass spectrometry (MS) has been available for decades, but improvements in the size, speed and cost have brought this technology to a point where it can be used for both broad-range and target-specific identification of pathogens. PCR-electrospray ionization mass spectrometry (PCR-ESI/MS) holds particular promise given that it can identify minute quantities and mixtures of nucleic acids from microbial isolates or directly from clinical specimens . A single process can, therefore, potentially identify bacterial, viral and fungal pathogens. Recently PCR-ECI/MS has been shown to be superior to culture for diagnosing prosthetic joint infections .
A different use of MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that is also a protein/peptide diagnostic tool, has been shown to have utility in identifying microorganisms at a species level. MALDI-TOF-MS has predominantly been assessed as a means of rapidly identifying the identity of both bacteria and their bacterial products from positive blood cultures, up to 24 h faster than conventional methods. A comparison study of PCR-ESI/MS and MALDI-TOF-MS suggested they have very similar performance characteristics in identifying organisms from positive blood cultures ; however, with rapidly evolving technology caution should be taken in over interpreting comparisons on platforms that are constantly being improved. One potential significant limitation of current MALDI-TOF-MS is that a large when a large mixture of bacteria are present, as occurs more commonly in HAP and VAP, the sensitivity and specificity become suboptimal .
Next-generation sequencing (NGS), also known as high-throughput sequencing, is a generic term used to describe a number of different modern sequencing technologies including Illumina (Solexa) sequencing, Roche 454 sequencing and Ion torrent, Proton/PGM sequencing and SOLiD sequencing. These recent technologies allow sequencing of DNA and RNA much more quickly and cheaply than the previously used Sanger sequencing. Developed initially for genetic mutation analysis, these platforms have potential uses in pathogen diagnosis. NGS has been particularly useful in diagnosing new and/or novel pathogens for which there are no available assays.
There is no doubt that our current empiric approach to treating CAP leads to significant overtreatment with antibiotics and equally that occasionally there are adverse outcomes due to unexpected pathogens. Moving to a pathogen-directed approach should therefore lead to better patient outcomes. Unfortunately to date our existing microbiological tests have failed to meet clinical need at multiple levels including speed, accuracy and ease of use.
New technology continues to develop at a rapid rate and a number of point-of-care platforms have the potential to significantly alter our approach to treating pneumonia. It is difficult currently to predict who the ‘winners’ will be, but speed of result delivery, ease of use and economics will all play a significant role in determining which will be broadly adopted. A summary of current and potential future tests against the criteria for an ideal test is provided in Table 1.
Several significant hurdles remain before any new platform will receive wide acceptance as a new standard of care. First, an exceptionally high, close to 100%, sensitivity and specificity will likely need to be demonstrated in a broad range of clinical settings before clinicians will feel confident in acting on the results. Secondly, even if they are confident in the results, significant changes in clinical practice will need to occur to make these platforms cost-effective, which will require major changes to current clinical practice as evidenced by the reluctance of clinicians to act on existing tests.
Financial support and sponsorship
G.W.W. has received financial support from the National Health and Medical Research Council of Australia.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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Also an excellent meta-analysis of urine testing for pneumococcal disease.
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An excellent review of the performance of rapid influenza diagnostic tests.
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Although in the setting of nosocomial pneumonia, this is the first of no doubt a series of papers analyzing this platform in the setting of respiratory tract infections.
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Keywords:Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
cause; diagnostics; empiric therapy; pneumonia