Breathing New Life into Pneumonia Diagnostics. Unit, Canterbury Health Laboratories, Christchurch, New Zealand; 3 Department of International

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1 JCM Accepts, published online ahead of print on 9 September 2009 J. Clin. Microbiol. doi: /jcm Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Breathing New Life into Pneumonia Diagnostics David R. Murdoch, 1,2 * Katherine L. O Brien, 3 J. Anthony G. Scott, 4,5 Ruth A. Karron, 3 Niranjan Bhat, 3,6 Amanda J. Driscoll, 3 Maria Deloria Knoll, 3 Orin S. Levine 3 1 Department of Pathology, University of Otago, Christchurch, New Zealand; 2 Microbiology Unit, Canterbury Health Laboratories, Christchurch, New Zealand; 3 Department of International Health, John Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; 4 KEMRI Wellcome Trust Research Programme, Kilifi, Kenya; 5 Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK; 6 Division of Infectious Diseases, Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, USA *Corresponding author: Department of Pathology, University of Otago, Christchurch, P.O. Box 4345, Christchurch, New Zealand; tel: ; fax: ; david.murdoch@cdhb.govt.nz Running title: pneumonia diagnostics

2 November 2, 2009 will mark the first World Pneumonia Day, launched by a coalition of child health organizations to support global efforts to prioritize pneumonia treatment and prevention. Despite recent medical advances, pneumonia takes the lives of up to 2 million children under the age of five each year more than AIDS, malaria and measles combined (15). As with many other illnesses, the disease burden is greatest among the world s most vulnerable population children living in developing nations. For every child who dies of pneumonia in a developed country, more than 2,000 die in developing countries (16). With safe and effective vaccines against Haemophilus influenzae type b (Hib), Streptococcus pneumoniae and measles, and with improvements in environment and nutrition, and in case management standards, we now have several strategies to both prevent and treat pneumonia. The challenge is to ensure universal access to these life-saving interventions. Laboratory diagnostics and clinical microbiologists should play an important part in global efforts to prevent and treat pneumonia. Diagnostic testing has an essential role in assuring the most appropriate and effective therapy for individual patients. It also plays a role in disease surveillance to define the etiologic spectrum of pneumonia cases and deaths. This in turn forms the evidence base for strategic decisions by global decision makers (such as the World Health Organization, UNICEF, and the GAVI Alliance), vaccine manufacturers, and funders to develop and support treatment algorithms, vaccine products and programs, and other effective prevention strategies. World Pneumonia Day also reminds us that much work remains to be done in pneumonia diagnostics, and that historically, even with the best of methods, we have been unable to define the microbial etiology of a significant proportion of pneumonia episodes, particularly in children. The conditions where pneumonia mortality is greatest are also the conditions where adequate diagnosis is least possible. Even in settings with access to state of the art microbiologic

3 diagnosis, establishing the etiology of a pneumonia case is fundamentally vexed by the limited ability to obtain specimens from the site of infection, without contamination by upper respiratory secretions. Lack of sensitive laboratory diagnostic tools has probably played a direct role in the delayed introduction of effective vaccines to prevent pneumonia. Vaccines against Hib and S. pneumoniae alone may prevent >50% of severe childhood pneumonia, but poor diagnostics contribute to substantial underdiagnosis and thus substantial efforts need to be put into place to build awareness about the potential impact of these interventions. In spite of these limitations, significant advances have been made in diagnostic technology. However, technology alone is not a panacea for pneumonia diagnostic needs: assessment of performance within clinical and epidemiologic contexts is essential. Laboratory diagnostics for pneumonia in 2009 The routine microbiological evaluation of patients with suspected pneumonia continues to rely on methods that have been around since Margaret Pittman s time: microscopy and culture of lower respiratory tract specimens, blood cultures, detection of antigens in urine, and serology. While there have been refinements to these traditional laboratory tools, they have led to only modest improvements in overall diagnostic capability. Indeed, some have argued that we are doing less well now in diagnosing the cause of pneumonia than we were doing 70 years ago (2). Recent advances in pneumonia diagnostics have most notably occurred in the areas of antigen and nucleic acid detection. Commercial antigen detection assays are now widely available for several pneumonia pathogens, particularly S. pneumoniae, Legionella pneumophila and some respiratory viruses. New generation immunochromatographic pneumococcal urinary antigen tests that detect the C-polysaccharide cell wall antigen have proven useful for diagnosing 3

4 pneumococcal pneumonia in adults (19). Detection of soluble Legionella antigen in urine is now well-established as a diagnostic tool for Legionnaires disease (6). Several commercial rapid tests, using immunochromatographic, ELISA or other formats, are now available for respiratory viruses including influenza and respiratory syncytial viruses (11, 12). The ease of performance of these kits has been responsible for their widespread use, including as near-patient tests. Nucleic acid detection tests (NATs), such as PCR, have been developed for all major pneumonia pathogens, although the development of commercial assays and their introduction into routine use by diagnostic laboratories have been relatively slow (7). NATs have many features that make them attractive tools for diagnosing the etiology of respiratory tract infections. They detect very low levels of nucleic acid from potentially all respiratory pathogens, do not depend on the viability of the target microbe, can provide results within a clinically-relevant time frame, are probably less affected by prior antibiotic administration than culture-based methods, and have the potential to provide supplementary information such as the presence of antibiotic resistance genes. NATs have been particularly useful for diagnosing infections that are difficult to rapidly diagnose by other methods (e.g. rhinovirus infection) and are now regarded as the preferred methods for detecting some respiratory pathogens, such as the respiratory viruses and Chlamydophila pneumoniae. Problems with pneumonia diagnostics Despite these technological advances, there are several major challenges that hinder the search for and understanding of the etiology of pneumonia Inability to obtain specimens from the site of infection. The detection of known pathogens in good quality specimens collected directly from the lower respiratory tract provides good 4

5 evidence of the likely microbial etiology of pneumonia, especially for microorganisms that do not normally colonize the upper respiratory tract. However, obtaining these specimens can be difficult creating a fundamental problem with pneumonia diagnostics. While efforts are directed towards the development of assays with ultra-high analytical sensitivity, the fact that an appropriate clinical specimen may not even be obtained from a patient in the first place is often overlooked in research activities. Even in adults with pneumonia, a large proportion do not produce sputum, including less than half of those with Legionnaires disease (6). Obtaining sputum from children is much more difficult and is usually only possible through induction procedures, which may in themselves cause concern about safety in children with respiratory distress. Difficulties obtaining sputum may prompt the use of more invasive techniques such as bronchoscopy and transthoracic needle aspiration (18). The latter is a relatively safe procedure if it is performed by trained staff on appropriately selected patients, and has the potential to define pneumonia etiology with high sensitivity and specificity. Further efforts to enhance the collection of specimens from the lower respiratory tract could make a major contribution to improving the identification of etiologic agents from pneumonia episodes. Difficulties establishing pneumonia causation. There are two particular challenges when testing respiratory specimens in the context of pneumonia. First, for a pneumonia pathogen that can colonize the upper airways of healthy individuals (e.g. S. pneumoniae), does detection in a lower respiratory specimen represent infection or contamination with a colonizing organism? Second, for a pathogen that replicates in (but does not colonize) the upper airways and causes a spectrum of diseases (e.g. rhinovirus), does detection in a respiratory specimen indicate that this organism is the cause of pneumonia or could this be a coincidental finding following a recent unrelated upper respiratory tract infection (URTI)? 5

6 Distinguishing colonization from infection is a major challenge when processing any sputum specimen. Quality checks based on the relative numbers of squamous epithelial cells and leukocytes seen in a sputum Gram stain smear may help detect upper airway contamination (8), especially if correlated with culture results. Despite the ongoing debate about the utility of sputum culture, there is at least some evidence that prompt examination of a high quality sputum specimen collected before antibiotic treatment has reasonably high sensitivity and specificity for pneumococcal pneumonia in adults (9). Lower respiratory tract specimens obtained by bronchoscopy can also be contaminated with upper airway flora, but the likelihood of contamination is generally regarded to be less. All the major viruses that cause pneumonia are more usually associated with non-pneumonic illnesses, particularly URTIs. Viral URTIs are common, especially in young children who typically experience >4 URTIs annually. With each episode, virus can typically be detected for at least 7 days, which means that the average child may test positive for a respiratory virus for at least one month of every year. Some viral URTIs occur at the same time as pneumonia and these may be causal (either directly or by leading to secondary bacterial pneumonia) or they may be incidental. In a study setting, these can be distinguished by determining the background prevalence of nasopharyngeal viral infection in a control group. Unfortunately, most studies of viral pneumonia have not used control groups and have therefore not questioned the implication that a positive test from a nasopharyngeal specimen indicates causation (5, 14). To further complicate matters, there is the problem of how to ascribe causality when more than one pathogen is detected within an individual; this is more likely to occur with use of multiple sensitive testing methods. 6

7 Distinguishing colonization from infection extends to the testing of non-respiratory specimens as well. Although newer generation pneumococcal urinary antigen tests have been an important advance in adults, they cannot be used reliably in children as they detect pneumococcal carriage in this age group (4). Poor clinical sensitivity. Most diagnostic tests for pneumonia pathogens have suboptimal diagnostic sensitivity. Blood cultures are frequently performed on hospitalized pneumonia patients, but are positive in <10% of cases. Blood cultures are also the main diagnostic tool used in surveillance programs for invasive pneumococcal disease, and are clearly only able to identify a minority of patients with pneumococcal pneumonia. Some fastidious pneumonia pathogens, such as Legionella spp. and C. pneumoniae, can be difficult to isolate even with use of special culture methods. Current commercial urinary antigen detection assays can only reliably detect infection caused by L. pneumophila serogroup 1, and not other species and serogroups. Serological testing may return falsely negative results if the convalescent serum is collected too early, before a detectable increase in antibodies, or not collected at all, or if acute serum is collected too late. While having the advantage of producing quick results, rapid antigen tests for influenza and respiratory syncytial virus are generally less sensitive than culture-based methods. Inadequate validation of new tests. Unfortunately, many new tests (particularly NATs) are introduced into diagnostic laboratories before adequate sensitivity and specificity data are generated. Before a test is introduced into a clinical diagnostic laboratory, it should first be thoroughly evaluated to determine analytical sensitivity and specificity. It should then be applied to different populations to determine clinical sensitivity and specificity, which is distinct from analytic sensitivity and specificity measures (10). If the test is to be applied to multiple specimen types, clinical sensitivity and specificity need to be determined for each specimen type. 7

8 All too often analytical sensitivity is confused with clinical (diagnostic) sensitivity (10). Analytical sensitivity (lower limit of detection) is the smallest amount of target that can be accurately detected by the assay, is relatively straightforward to calculate in laboratory-based studies, and is expressed as a concentration. Clinical sensitivity is the proportion of true positive patient samples that are correctly identified by the assay, and is a measure of test validity in a population. Its measurement involves testing clinical specimens and comparing the new test with a gold standard test; it is expressed as a proportion or percentage. In spite of these important differences, the two terms are frequently used interchangeably in clinical settings. Very high analytical sensitivity is often used to promote NATs, despite the fact that high analytical sensitivity does not guarantee high clinical sensitivity (e.g. as occurs when blood-based inhibitors of Taq polymerase are not removed prior to annealing). Although it is desirable for an assay to have a high analytical sensitivity, the biological relevance beyond a certain point may be minimal. An assay that is so sensitive that it detects harmless asymptomatic infection may lead to difficulties in attributing etiology to a pathogen because of the frequency with which it is found among control subjects. In these circumstances a quantitative approach may be preferable. Another difficulty is the accurate calculation of clinical specificity due to the lack of suitable gold standards. This is an inherent problem with all NATs, both within and outside the context of pneumonia. NATs are likely to have higher clinical sensitivity than most traditional diagnostic tools. Consequently, a positive NAT result cannot necessarily be dismissed as being falsely positive simply because a less sensitive comparison test is negative Tests with high clinical sensitivity and specificity do not entirely address the issue of causality. A pathogen may truly be present in a patient as evidenced by a reliable assay, but that does not, for every pathogen allow us to ascribe a given episode of disease to that pathogen. Epidemiologic 8

9 sensitivity and specificity, which can only be established from population based studies which include control subjects, and employ a panel of highly sensitive and specific assays, inherently includes the analytic, clinical and causal components of pathogen detection. Vaccine probe studies which uncover the attributable burden of a given pathogen are another approach to firmly ascribing the proportion of pneumonia to one or more pathogens. Future directions To advance pneumonia diagnostics we need to improve current technology and simultaneously explore innovative approaches. We need to give equal attention to the specimen type and how we collect it as we do to laboratory assay development. New diagnostics must be systematically and rigorously evaluated for analytic, clinical and eventually epidemiologic sensitivity and specificity. Strategies to determine the etiology of pneumonia in children are a special priority. NATs have now been developed to a stage whereby multiplex assays that detect all common respiratory pathogens are commercially available in user friendly platforms, and further improvements in design and performance are expected. The emphasis should now be placed on clarifying the clinical usefulness of these assays, developing standardized methods, producing even more user-friendly platforms, and exploring the role of quantitative assays. Antigen detection assays in immunochromatographic or similar formats are fast and simple to perform. In many ways these methods are among the most attractive diagnostic tools, as illustrated by the success of rapid diagnostic tests for malaria (17), but further development is dependent on the discovery of suitable antigens that can be reliably detected in readily obtained samples Breath analysis is an example of an innovative area with enormous potential for pneumonia diagnostics. Bacteria and fungi produce volatile metabolites that may be used as biomarkers (1). 9

10 Detection of these biomarkers in breath samples by gas chromatography/mass spectroscopy (GC/MS) or similar methods may provide an etiological diagnosis of pneumonia. Potential biomarkers have been reported for some respiratory pathogens (3, 13), but it is still uncertain whether they will prove to be useful as clinical diagnostic tools. Regardless, this sort of innovation is welcome in the world of pneumonia diagnostics. Lastly, it is important to remember that the greatest burden of pneumonia is in developing countries. To enable deployment in under-resourced settings, laboratory diagnostic tests for pneumonia need to be rapid, inexpensive, easy to use, and should be useful for surveillance purposes. However, there is also a role for more expensive high-tech tests that may not require wide availability. There are good reasons to determine the causes of pneumonia which may be best satisfied by deployment of sophisticated tests in sentinel sites, viz. (i) development of empiric treatment guidelines in the world following the introduction of vaccines against Hib and S. pneumoniae, and (ii) evaluation of the impact of vaccines (and other preventive interventions) on the incidence of pneumonia. As the spectrum of pneumonia causing pathogens changes because of vaccine introduction, antimicrobial resistance, HIV/AIDS, urbanization, and socioeconomic changes, our ability to prevent and treat this life-threatening syndrome is anchored in our ability to monitor the etiologic spectrum of disease-causing pathogens. Significant efforts will be required in these settings to build and sustain the capacity of local laboratories and laboratorians, and to link laboratory findings with clinical management of individual patients. World Pneumonia Day provides a useful reminder that much more can and must be done to improve the diagnosis, treatment, and prevention of pneumonia worldwide, and that clinical microbiologists play an important role in the process. 10

11 Acknowledgments This work was performed under the Pneumonia Etiology Research for Child Health (PERCH) program at the Johns Hopkins Bloomberg School of Public Health and was funded in full by a grant from The Bill & Melinda Gates Foundation. NB is supported by grant 1KL2RR from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at Information on Re-engineering the Clinical Research Enterprise can be obtained from Downloaded from on November 19, 2018 by guest 11

12 References 1. Allardyce, R. A., V. S. Langford, A. L. Hill, and D. R. Murdoch Detection of volatile metabolites produced by bacterial growth in blood culture media by selected ion flow tube mass spectrometry (SIFT-MS). J. Microbiol. Meth. 65: Bartlett, J. G Decline in microbial studies for patients with pulmonary infections. Clin. Infect. Dis. 39: Chambers, S. T., M. Syhre, D. R. Murdoch, F. McCartin, and M. J. Epton Detection of 2-Pentylfuran in the breath of patients with Aspergillus fumigatus. Med. Mycol. 47: Dowell, S. F., R. L. Garman, G. Liu, O. S. Levine, and Y.-H. Yang Evaluation of Binax NOW, an assay for the detection of pneumococcal antigen in urine samples, performed among pediatric patients. Clin. Infect. Dis. 32: Jennings, L. C., T. P. Anderson, K. A. Beynon, A. Chua, R. T. R. Laing, A. M. Werno, S. A. Young, S. T. Chambers, and D. R. Murdoch Incidence and characteristics of viral community-acquired pneumonia in adults. Thorax 63: Murdoch, D. R Diagnosis of Legionella infection. Clin. Infect. Dis. 36: Murdoch, D. R Molecular genetic methods in the diagnosis of lower respiratory tract infections. APMIS 112: Murray, P. R., and J. A. I. Washington Microscopic and bacteriologic analysis of expectorated sputum. Mayo Clin. Proc. 50: Musher, D. M., R. Montoya, and A. Wanahita Diagnostic value of microscopic examination of gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia. Clin. Infect. Dis. 39:

13 10. Saah, A. J., and D. R. Hoover "Sensitivity" and "specificity" reconsidered: the meaning of these terms in analytical and diagnostic settings. Ann. Intern. Med. 126: Selvarangan, R., D. Abel, and M. Hamilton Comparison of BD Directigen EZ RSV and Binax NOW RSV tests for rapid detection of respiratory syncytial virus from nasopharyngeal aspirates in a pediatric population. Diagn. Microbiol. Infect. Dis. 62: Smit, M., K. A. Beynon, D. R. Murdoch, and L. C. Jennings Comparison of the NOW Influenza A & B, NOW Flu A, NOW Flu B, and Directigen Flu A+B assays, and immunofluorescence with viral culture for the detection of influenza A and B viruses. Diagn. Microbiol. Infect. Dis. 57: Syhre, M., L. Manning, S. Phuanukoonnon, P. Harino, and S. T. Chambers The scent of Mycobacterium tuberculosis Part II breath. Tuberculosis 89: Tsolia, M. N., S. Psarras, A. Bossios, H. Audi, M. Paldanius, D. Gourgiotis, K. Kallergi, D. A. Kafetzis, A. Constantopoulos, and N. G. Papadopoulos Etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections. Clin. Infect. Dis. 39: UNICEF Pneumonia: the forgotten killer of children. UNICEF/WHO, Geneva. 16. UNICEF Progress for children: a world fit for children statistical review. UNICEF, NY. 17. UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Malaria rapid diagnostic test performance: results of WHO product testing of malaria RDTs: round 1 (2008). World Health Organisation, Geneva. 13

14 18. Vuori-Holopainen, E., E. Salo, H. Saxén, K. Hedman, T. Hyypiä, R. Lahdenperä, M. Leinonen, E. Tarkka, M. Vaara, and H. Peltola Etiological diagnosis of childhood pneumonia by use of transthoracic needle aspiration and modern microbiological methods. Clin. Infect. Dis. 34: Werno, A. M., and D. R. Murdoch Laboratory diagnosis of invasive pneumococcal disease. Clin. Infect. Dis. 46: Downloaded from on November 19, 2018 by guest 14

Diagnosis of Pneumococcal Disease

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