- Email: [email protected]
Rapid detection of Streptococcus pneumoniae in community-acquired pneumonia
REVIEW Rapid detection of Streptococcus pneumoniae in community-acquired pneumonia T. M. File Jr1 and R. S. Kozlov2 1
Summa Health System, Akron, OH, and North-eastern Ohio Universities College of Medicine, Rootstown, OH, USA and 2Institute of Antimicrobial Chemotherapy, Smolensk State Medical Academy, Smolensk, Russia
ABSTRACT The utility of diagnostic studies to determine the aetiology of community-acquired pneumonia is controversial because of the lack of rapid, easily performed, accurate, cost-effective methods that provide immediate results for the majority of patients at the initial evaluation by a clinician in an office or acute-care setting. Nevertheless, there are good reasons for establishing an aetiological diagnosis, namely to direct antibiotic management for an individual patient, to facilitate management of treatment failure and, by de-escalation or narrowing of antibiotic therapy, to potentially decrease healthcare costs, drug side-effects and the selection pressure for antibiotic resistance. This article outlines the clinical value of Gram’s stain and briefly surveys current tests for Streptococcus pneumoniae based on immunological and molecular assays. Keywords
Pneumococcus, pneumonia, rapid detection
Clin Microbiol Infect 2006; 12 (suppl 9): 27–33
INTRODUCTION Although numerous pathogens can cause community-acquired pneumonia (CAP), Streptococcus pneumoniae remains the leading bacterial cause and the leading cause of mortality [1]. It is essential that initial empirical therapy of CAP be effective against this important pathogen. However, the empirical therapy of CAP has become more complicated over the past several decades, owing to the emergence of newly recognised pathogens (i.e., several ‘atypical’ pathogens and viruses) as well as the increasing emergence of pneumococcal strains resistant to penicillin and other antimicrobial agents. While the clinical relevance of much of the in-vitro resistance in S. pneumoniae is questionable, the presence of high-level resistance (e.g., a penicillin MIC >4 mg ⁄ L) does seem to affect treatment outcomes [2]. In addition, resistant pneumococcal infection in patients who require hospitalisation is associated with increased length of stay and cost of care [2]. Corresponding author and reprint requests: T. M. File, Jr, Suite 105, 75 Arch St, Akron, Ohio 44304, USA E-mail: [email protected]
In light of these concerns regarding the empirical therapy of CAP, it is important to examine the potential impact of the rapid diagnosis of S. pneumoniae as the cause of an infection in a specific patient. This article reviews the available tests that allow the rapid detection of S. pneumoniae— namely Gram’s stain and immunological and molecular assays—and discusses their clinical usefulness in directing antimicrobial therapy. GRAM’S STAIN The value of routinely performing a Gram stain of the sputum and culture has long been debated. These tests are limited by the fact that many patients cannot produce a good specimen, patients often receive antimicrobial agents prior to evaluation, and many specimens give inconclusive results. The validity of Gram’s stain is related directly to the experience of the interpreter. Indeed, some discrepant findings concerning the Gram stain of the sputum are presumably explained by the quality of specimens and the technical expertise of medical staff. When stringent criteria are applied, although the sensitivity drops, the specificity for pneumococcal pneumonia can
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approach 90%. For example, the yield of S. pneumoniae is only 40–50% from sputum cultures obtained from patients with bacteraemic pneumococcal pneumonia in studies done a few decades ago [3,4]. A more recent study of 100 bacteraemic pneumococcal pneumonias found that sputum specimens were not submitted in 31% of cases and were judged as inadequate in another 16% of cases [5]. If patients receiving antibiotics for >24 h were excluded, Gram’s stain showed S. pneumoniae in 63% of cases and the culture was positive in 86%. For patients who had not received antibiotics, Gram’s stain was read as being consistent with S. pneumoniae in 80% of cases and sputum culture was positive in 93%. While favourable reports of Gram’s stain have been published, a meta-analysis showed a low yield in terms of the number of patients with adequate specimens and definitive results [6]. At least 40% of patients are unable to produce any sputum or sputum in a timely manner, and while the yield of cultures is substantially higher with endotracheal aspirates, bronchoscopic samples or trans-thoracic needle aspirates, specimens obtained after initiation of antibiotic therapy are unreliable and must be interpreted carefully. A final limitation of the sputum Gram’s stain, especially as applied in the USA, is the notable decline in its use by house staff and attending physicians when initially evaluating patients with pneumonia. This is in part due to the Clinical Laboratory Improvement Act of 1988, which required that staff have credentials to interpret Gram’s stains of any specimens. In addition, outsourcing of specimens to laboratories outside the hospital leads to delays in processing and less direct communication between the microbiology laboratory and the clinician. IMMUNOLOGICAL ASSAYS Immunological assays are the most commonly used rapid assays for S. pneumoniae. Tests that fall into this category include counter-immunoelectrophoresis, co-agglutination, latex agglutination, ELISA, immunofluorescence and immunochromatography. Counter-immunoelectrophoresis Counter-immunoelectrophoresis is a relatively old and labour-intensive technique. Its sensitivity in
diagnosing pneumococcal pneumonia by testing serum, urine and bronchoalveolar lavage fluid has been shown to be low (not exceeding 50%) [7,8]. However, the technique is able to identify S. pneumoniae in blood cultures at least 24 h earlier than is possible using subculturing, with almost 100% sensitivity and specificity [9]. In the same manner, agglutination tests have proved useful for the rapid and specific identification of pneumococcal antigen blood culture bottles and for the prevention of false-negative culture results caused by autolysis of pneumococci during incubation. Co-agglutination and latex agglutination Pneumolysin-based co-agglutination and C-substance-specific latex agglutination are inexpensive, easy to perform and to read, and are relatively independent of antibiotic therapy. These methods can be used for aetiological diagnosis of pneumonia and meningitis by the detection of pneumococcal antigens in sputum, bronchoalveolar and pleural fluid, serum, urine, blood culture broth and cerebrospinal fluid. They are used mostly in laboratory settings, although commercial kits are also available. Serum co-agglutination tests show a sensitivity of c. 90% in diagnosing pneumonia, while cerebrospinal fluid tests for meningitis show a sensitivity of c. 71–97% [10,11]. The performance of latex agglutination methods varies considerably, depending upon the clinical specimen. Latex agglutination had low sensitivity in detecting pneumococcal antigen in serum (18–47%) and urine (14–29%) [7], but was found to be very sensitive when testing sputum (90–94%) and blood cultures (100%) [12,13]. At the same time, latex agglutination is characterised by high specificity. The technique is a frequently performed laboratory procedure, but its clinical utility is controversial. A review of 5169 assays (for S. pneumoniae, group B streptococcus, Haemophilus influenzae and Neisseria meningitidis) performed over a 10-month period at two sites showed that only 57 (1.1%) of tests were positive, of which 31 (54%) were false-positives [14]. Therapy was not altered on the basis of any of the true-positive results, but false-positive results led to additional costs, prolonged hospitalisation and some clinical complications. Thus, this retrospective study did not support the use of latex agglutination for rapid antigen detection.
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Elisa ELISA-based rapid tests allow a diagnosis of pneumococcal pneumonia to be made with a sensitivity of >70% by the detection of capsular antigen in serum [15] or pleural fluid [16]. However, this method is not widely used and is not yet commercially available. Immunochromatography Immunochromatography is a modern and frequently used technique that can be used to determine the aetiology of pneumonia, meningitis and otitis media, to establish the diagnosis of invasive pneumococcal disease, and for the documentation of nasopharyngeal colonisation with S. pneumoniae. Several commercial kits based on immunochromatography are currently available. This technique is suitable for the identification of pneumococci in different types of clinical material, as well as for the detection of antigenuria in patients with pneumococcal diseases. Urinary antigen tests are 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 bacteraemic cases, the pneumococcal urinary antigen detected by immunochromatographic assay was still positive in 83% of cases after 3 days of therapy [17]. This form of urinary antigen testing has the principal additional advantages of rapidity (c. 15 min), simplicity and reasonable specificity in adults. Studies in adults have shown a sensitivity of 50–80% and specificity exceeding 90% [18,19]. The immunochromatography assay is also highly accurate in diagnosing pneumococcal meningitis (95% sensitivity with cerebrospinal fluid, 57% sensitivity with urine, and 100% specificity) [20]. The disadvantages of urinary antigen testing include the cost and the lack of an isolated organism for in-vitro susceptibility tests. Notably, immunochromatography is not suitable for evaluation of therapeutic effect, because positive test results are obtained for several weeks after recovery. Moreover, the immunochromatographic assays are non-specific for pneumococcal infections in children, particularly the very young, since nasopharyngeal carriage of S. pneumoniae can cause false-positive results [21]. Likewise, the
test may not be useful for predicting the aetiology of disease in populations with a high rate of pneumococcal nasopharyngeal carriage. Falsepositive results have been seen in patients with a prior episode of CAP within the previous 3 months, but they do not appear to be a significant problem in colonised patients with chronic obstructive pulmonary disease [18]. Recent studies have evaluated the clinical utility of urinary antigen testing for S. pneumoniae in patients with CAP. Guchev et al. [22] prospectively assigned patients with mild pneumonia to two groups. Those with positive urinary antigen test results were treated using pneumococcaldirected therapy with amoxycillin. Those with negative urinary antigen test results were treated with clarithromycin, based on perceived likelihood of infection by atypical pathogens. Of 219 evaluable patients, 22% had a positive urinary antigen test result. There was no difference in the outcomes of the two groups. Notably, 47 ⁄ 71 (62%) of patients in whom an atypical pathogen was identified were in the urinary antigen-negative arm, while 24 (38%) were in the urinary antigenpositive arm, indicating that they had S. pneumoniae in association with an atypical pathogen. However, since the atypical pathogens were determined by serological methods, these latter cases might have represented a primary atypical infection followed by secondary infection by S. pneumoniae. In such cases, it is probable that the clinical manifestations of infection that were treated were attributable to S. pneumoniae, and this may explain the good response to amoxycillin alone. The authors concluded that the urinary antigen test allowed them to administer targeted therapy with a penicillin-class antibiotic rather than a broader-spectrum agent. Such narrow-spectrum therapy can be more cost-effective and can allow broad-spectrum agents, such as macrolides or fluoroquinolones, to be reserved for patients whose urinary antigen test result is negative. Potential cost reductions are likely to be influenced by price differences between the targeted and broad-spectrum agents and by the proportion of positive test results [23]. Also, it should be noted that at the time of this study, in Russia, there was little b-lactam or macrolide resistance in S. pneumoniae. Thus, this approach may not be as useful in areas where high-level penicillin resistance exists. In addition, the study was performed
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in military trainees who were young (mean age, 19 years) and generally healthy. The authors suggested that additional trials are necessary for other clinical settings [22]. In another study, Stralin and Holmberg evaluated the urinary antigen test in 215 hospitalised patients with CAP, all of whom received initial b-lactam monotherapy [24]. The median age was 74 years, and approximately 45% of patients had a pneumonia severity index of class IV or V. Thus, these patients were more severely ill than those in the previously described study [22]. Thirty-eight patients had a positive urinary antigen result for S. pneumoniae, and 92% of these had a successful outcome; 114 had a negative urinary antigen result, and 78% of these had a successful outcome. There were no patients with a positive PCR sputum test result for an atypical pathogen in the urinary antigen-positive group, whereas six patients had a positive PCR result for Mycoplasma spp. or Chlamydophila spp. in the urinary antigennegative group. The authors suggested that a positive urinary antigen test result supports treatment with narrow-spectrum b-lactam antibiotics and that additional coverage for atypical pathogens is needed more frequently in patients with negative test results. MOLECULAR ASSAYS Molecular assays for S. pneumoniae include nucleic acid amplification techniques, i.e., PCR and loop-mediated isothermal amplification, and DNA hybridisation. PCR is a potentially attractive diagnostic tool for rapid diagnosis, since it does not rely on bacterial growth or the viability of the organism. There is some evidence that PCR-based assays are more sensitive than culture. PCR assays show moderate sensitivity (43%) in tests of blood, and high specificity in blood-culture-confirmed pneumococcal infections, and sometimes give positive results in culture-negative samples [25]. PCR techniques based on amplification of the pneumolysin or autolysin genes are applicable for the diagnosis of pneumonia, otitis media and meningitis. In sputum tests, autolysin and pneumolysin PCR has shown a high sensitivity (more than 80%), but a low specificity (30–40%) [26]. However, in pleural fluid PCR detects the pneumolysin gene with a sensitivity of 78% and a specificity of 93% [27].
The interpretation of sputum PCR tests is complicated by the difficulty in differentiating between pneumococcal colonisation and true infection. One approach that may help to resolve this problem is the quantification of the target organism by real-time PCR. Yang et al. evaluated the utility of a real-time pneumolysin gene PCR test using sputum samples from patients admitted with CAP [28]. Of 129 patients, 23% had S. pneumoniae isolated from blood or sputum. The sensitivity and specificity using real-time PCR were 90% and 80%, respectively. Real-time PCR can also detect S. pneumoniae in middle ear fluid from children with acute otitis media with a sensitivity of 100% and a specificity of 73% compared to culture [29]. Multiplex PCR allows the identification of several pathogens simultaneously in samples from patients with pneumonia, meningitis and otitis media with high levels of accuracy [30]. Loop-mediated isothermal amplification is a novel nucleic acid amplification technique that amplifies DNA under isothermal conditions (63C) with high specificity, efficiency and rapidity. It is characterised by a sensitivity 1000 times greater than that of conventional PCR [31]. PCR may therefore represent a useful diagnostic adjunct to assist clinicians in the selection of the most appropriate antibiotics. At present, conventional culture methods will still be required to confirm the antimicrobial susceptibility of the organism. However, PCR techniques also have the potential to be used for the rapid detection of antimicrobial resistance in S. pneumoniae. Amplification of the pneumococcal penicillin-binding protein 2B gene can be used to determine penicillin susceptibility, and provides susceptibility data even in culture-negative cases. The method has positive and negative predictive values of 100% and 91%, respectively, and showed 98.3% agreement with MIC data [32]. DISCUSSION The utility of diagnostic studies to determine the aetiology of CAP is controversial because of the lack of rapid, easily performed, accurate, costeffective methods that provide immediate results for the majority of patients at the point of service (i.e., the initial evaluation by a clinician in an office or acute-care setting). Nevertheless, there is a good rationale for establishing an aetiological
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File and Kozlov Rapid detection of Streptococcus pneumoniae 31
diagnosis. The primary reason is to provide results that influence the antibiotic management of an individual patient. The selection of antibiotic therapy can be broadened, narrowed or completely changed on the basis of accurate diagnostic testing. Furthermore, management of early antibiotic failure can be greatly facilitated by the determination of an aetiological diagnosis. De-escalation or narrowing of antibiotic therapy on the basis of diagnostic testing may decrease costs, drug side-effects and the selection pressure for antibiotic resistance. These considerations are important in light of the specific impact of the rapid diagnosis of S. pneumoniae. Since all recommended antimicrobial options for empirical therapy in most of the published guidelines are effective against S. pneumoniae (including drug-resistant strains), it can be argued that there is little need to identify S. pneumoniae in order to ensure appropriate therapy for this pathogen. In addition, the possibility of polymicrobial CAP and the potential benefit of combination therapy for bacteraemic pneumococcal pneumonia have complicated the decision to narrow antibiotic therapy, since the identification of S. pneumoniae may not preclude a second pathogen. Furthermore, one randomised clinical trial of a diagnostic strategy in CAP demonstrated that, while there was a significant increase in diagnostic yield, there were no statistically significant differences in mortality or length of stay between pathogen-directed therapy and empirical therapy [33]. However, this study did not require the alteration or specification of therapy on the basis of the results of the diagnostic tests. Moreover, the study was performed in a country with a low incidence of antibiotic resistance (The Netherlands), which may limit its applicability to areas with higher levels of resistance. It is of interest that pathogen-directed therapy was associated in this study with a lower mortality rate in the small number of patients admitted to the intensive care unit. Adverse effects of treatment were significantly more frequent in the empirical therapy group, but may be unique to the specific antibiotic used (erythromycin). The absence of benefit overall in this study should not be interpreted as an absence of benefit for an individual patient. The identification of S. pneumoniae can be helpful in de-escalating antimicrobial therapy, a matter of particular importance in this era of antimicrobial resistance. Hopefully, a reduction
in antimicrobial use will lead to reduced pressure for emergence of resistance, as well as a reduction in adverse events. Knowledge of the causative pathogen can also contribute to the rational selection of antimicrobial agents when changing from intravenous to oral therapy in the hospital setting. Finally, regarding the issue of combination therapy for bacteraemic pneumococcal pneumonia, it is important to note that these studies evaluated the effects of initial empirical therapy before the results of blood cultures were known and did not examine the effects of pathogenspecific therapy after the results of blood cultures were available [34]. The benefit of combination therapy was also most pronounced in the more severely ill patients. Therefore, no data suggest that discontinuation of combination therapy, once results of cultures are known, is unsafe in non-intensive-care-unit patients. Thus, if an appropriate culture reveals the presence of penicillin-susceptible S. pneumoniae, a pathogenspecific, narrow-spectrum agent (e.g., penicillin or amoxycillin) can be selected, with the aforementioned potential benefits with respect to the selective pressure for resistance. CONCLUSION The rapid detection of S. pneumoniae provides a unique opportunity for more targeted use of narrow-spectrum antibiotics, thus creating the possibility of more rational antibiotic use. Further studies on the clinical utility of existing and newer technologies should provide the evidence required for the integration of rapid tests and early pathogen-directed therapy into routine clinical practice. The rapid diagnosis of S. pneumoniae for CAP will require a different approach to clinical management than is presently followed in many countries. Presently, the general standard of care for CAP is empirical therapy, with little emphasis on microbiological identification. However, we may be entering an era in which aetiological diagnosis will become paramount [35]. This is stated in reference to the identification of pathogens other than S. pneumoniae (e.g., communityonset methicillin-resistant Staphylococcus aureus and H5N1 Influenza A virus), which are not effectively treated by most recommended empirical antimicrobial regimens.
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REFERENCES 1. File TM Jr. Community-acquired pneumonia. Lancet 2003; 362: 1991–2001. 2. File TM Jr. Streptococcus pneumoniae and community-acquired pneumonia: a cause for concern. Am J Med 2004; 117: 39S–50S. 3. Lentino JR, Lucks DA. Nonvalue of sputum culture in the management of lower respiratory tract infections. J Clin Microbiol 1987; 25: 758–762. 4. Garcia-Vazquez E, Marcos MA, Mensa J et al. Assessment of the usefulness of sputum culture for diagnosis of community-acquired pneumonia using the PORT predictive scoring system. Arch Intern Med 2004; 164: 1807–1811. 5. Musher DM, Montoya R, Wanahita A. Diagnostic value of microscopic examination of Gram-stained sputum and sputum cultures in patients with bacteremic pneumococcal pneumonia. Clin Infect Dis 2004; 39: 165–169. 6. Reed WW, Byrd GS, Gates RH Jr, Howard RS, Weaver MJ. Sputum gram’s stain in community-acquired pneumococcal pneumonia. A meta-analysis. West J Med 1996; 165: 197–204. 7. Lenthe-Eboa S, Brighouse G, Auckenthaler R et al. Comparison of immunological methods for diagnosis of pneumococcal pneumonia in biological fluids. Eur J Clin Microbiol 1987; 6: 28–34. 8. Jimenez P, Meneses M, Saldias F, Velasquez M. Pneumococcal antigen detection in bronchoalveolar lavage fluid from patients with pneumonia. Thorax 1994; 49: 872–874. 9. Waters C, Fossieck B Jr, Parker RH. Counterimmunoelectrophoresis for rapid identification of blood-culture isolates. Am J Clin Pathol 1979; 71: 330–332. 10. Mayer ME, Geiseler PJ, Harris B. Coagglutination for detection and serotyping of bacterial antigens: usefulness in acute pneumonias. Diagn Microbiol Infect Dis 1983; 1: 277–285. 11. Kanungo R, Bhaskar M, Kumar A, Badrinath S, Rajalakshmi B. Detection of pneumolysin in cerebrospinal fluid for rapid diagnosis of pneumococcal meningitis. Indian J Med Res 2004; 119: 75–78. 12. Wellstood S. Evaluation of a latex test for rapid detection of pneumococcal antigens in sputum. Eur J Clin Microbiol Infect Dis 1992; 11: 448–451. 13. Jesudason MV, Sridharan G, Arulselvan K, Joseph A, Steinhoff MC, John TJ. C substance-specific latex agglutination for early and rapid detection of Streptococcus pneumoniae in blood cultures. Indian J Med Res 1995; 102: 258–260. 14. Perkins MD, Mirrett S, Reller LB. Rapid bacterial antigen detection is not clinically useful. J Clin Microbiol 1995; 33: 1486–1491. 15. Mata M, Sesma P, Bernardez I. Usefulness of an ELISA for the diagnosis of Streptococcus pneumoniae in patients with pneumonia. Enferm Infecc Microbiol Clin 1996; 14: 255–257 [in Spanish]. 16. Requejo HI, Alkmin M das G, Almeida RG et al. Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples. Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination. Rev Inst Med Trop Sao Paulo 1994; 36: 531–537. 17. Smith MD, Derrington P, Evans R et al. Rapid diagnosis of bacteremic pneumococcal infections in adults by using the
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targeting the lytA gene for detection of Streptococcus pneumoniae. J Clin Microbiol 2005; 43: 1581–1586. 32. du Plessis M, Smith AM, Klugman KP. Application of pbp1A PCR in identification of penicillin-resistant Streptococcus pneumoniae. J Clin Microbiol 1999; 37: 628–632. 33. Van der Eerden Vlaspolder F, de Graaff CS, Grout T, Jansen HM, Boersma WG. Value of intensive diagnostic microbiological investigation in low- and high-risk
patients with community-acquired pneumonia. Eur J Clin Microbiol Infect Dis 2005; 24: 241–249. 34. File TM, Mandell LA. What is optimal antimicrobial therapy for bacteremic pneumococcal pneumonia. Clin Infect Dis 2003; 36: 396–398. 35. Bartlett JG. Decline in microbial studies for patients with pulmonary infections. Clin Infect Dis 2004; 39: 170–172.
2006 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 12 (suppl 9), 27–33
Summa Health System, Akron, OH, and North-eastern Ohio Universities College of Medicine, Rootstown, OH, USA and 2Institute of Antimicrobial Chemotherapy, Smolensk State Medical Academy, Smolensk, Russia
ABSTRACT The utility of diagnostic studies to determine the aetiology of community-acquired pneumonia is controversial because of the lack of rapid, easily performed, accurate, cost-effective methods that provide immediate results for the majority of patients at the initial evaluation by a clinician in an office or acute-care setting. Nevertheless, there are good reasons for establishing an aetiological diagnosis, namely to direct antibiotic management for an individual patient, to facilitate management of treatment failure and, by de-escalation or narrowing of antibiotic therapy, to potentially decrease healthcare costs, drug side-effects and the selection pressure for antibiotic resistance. This article outlines the clinical value of Gram’s stain and briefly surveys current tests for Streptococcus pneumoniae based on immunological and molecular assays. Keywords
Pneumococcus, pneumonia, rapid detection
Clin Microbiol Infect 2006; 12 (suppl 9): 27–33
INTRODUCTION Although numerous pathogens can cause community-acquired pneumonia (CAP), Streptococcus pneumoniae remains the leading bacterial cause and the leading cause of mortality [1]. It is essential that initial empirical therapy of CAP be effective against this important pathogen. However, the empirical therapy of CAP has become more complicated over the past several decades, owing to the emergence of newly recognised pathogens (i.e., several ‘atypical’ pathogens and viruses) as well as the increasing emergence of pneumococcal strains resistant to penicillin and other antimicrobial agents. While the clinical relevance of much of the in-vitro resistance in S. pneumoniae is questionable, the presence of high-level resistance (e.g., a penicillin MIC >4 mg ⁄ L) does seem to affect treatment outcomes [2]. In addition, resistant pneumococcal infection in patients who require hospitalisation is associated with increased length of stay and cost of care [2]. Corresponding author and reprint requests: T. M. File, Jr, Suite 105, 75 Arch St, Akron, Ohio 44304, USA E-mail: [email protected]
In light of these concerns regarding the empirical therapy of CAP, it is important to examine the potential impact of the rapid diagnosis of S. pneumoniae as the cause of an infection in a specific patient. This article reviews the available tests that allow the rapid detection of S. pneumoniae— namely Gram’s stain and immunological and molecular assays—and discusses their clinical usefulness in directing antimicrobial therapy. GRAM’S STAIN The value of routinely performing a Gram stain of the sputum and culture has long been debated. These tests are limited by the fact that many patients cannot produce a good specimen, patients often receive antimicrobial agents prior to evaluation, and many specimens give inconclusive results. The validity of Gram’s stain is related directly to the experience of the interpreter. Indeed, some discrepant findings concerning the Gram stain of the sputum are presumably explained by the quality of specimens and the technical expertise of medical staff. When stringent criteria are applied, although the sensitivity drops, the specificity for pneumococcal pneumonia can
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approach 90%. For example, the yield of S. pneumoniae is only 40–50% from sputum cultures obtained from patients with bacteraemic pneumococcal pneumonia in studies done a few decades ago [3,4]. A more recent study of 100 bacteraemic pneumococcal pneumonias found that sputum specimens were not submitted in 31% of cases and were judged as inadequate in another 16% of cases [5]. If patients receiving antibiotics for >24 h were excluded, Gram’s stain showed S. pneumoniae in 63% of cases and the culture was positive in 86%. For patients who had not received antibiotics, Gram’s stain was read as being consistent with S. pneumoniae in 80% of cases and sputum culture was positive in 93%. While favourable reports of Gram’s stain have been published, a meta-analysis showed a low yield in terms of the number of patients with adequate specimens and definitive results [6]. At least 40% of patients are unable to produce any sputum or sputum in a timely manner, and while the yield of cultures is substantially higher with endotracheal aspirates, bronchoscopic samples or trans-thoracic needle aspirates, specimens obtained after initiation of antibiotic therapy are unreliable and must be interpreted carefully. A final limitation of the sputum Gram’s stain, especially as applied in the USA, is the notable decline in its use by house staff and attending physicians when initially evaluating patients with pneumonia. This is in part due to the Clinical Laboratory Improvement Act of 1988, which required that staff have credentials to interpret Gram’s stains of any specimens. In addition, outsourcing of specimens to laboratories outside the hospital leads to delays in processing and less direct communication between the microbiology laboratory and the clinician. IMMUNOLOGICAL ASSAYS Immunological assays are the most commonly used rapid assays for S. pneumoniae. Tests that fall into this category include counter-immunoelectrophoresis, co-agglutination, latex agglutination, ELISA, immunofluorescence and immunochromatography. Counter-immunoelectrophoresis Counter-immunoelectrophoresis is a relatively old and labour-intensive technique. Its sensitivity in
diagnosing pneumococcal pneumonia by testing serum, urine and bronchoalveolar lavage fluid has been shown to be low (not exceeding 50%) [7,8]. However, the technique is able to identify S. pneumoniae in blood cultures at least 24 h earlier than is possible using subculturing, with almost 100% sensitivity and specificity [9]. In the same manner, agglutination tests have proved useful for the rapid and specific identification of pneumococcal antigen blood culture bottles and for the prevention of false-negative culture results caused by autolysis of pneumococci during incubation. Co-agglutination and latex agglutination Pneumolysin-based co-agglutination and C-substance-specific latex agglutination are inexpensive, easy to perform and to read, and are relatively independent of antibiotic therapy. These methods can be used for aetiological diagnosis of pneumonia and meningitis by the detection of pneumococcal antigens in sputum, bronchoalveolar and pleural fluid, serum, urine, blood culture broth and cerebrospinal fluid. They are used mostly in laboratory settings, although commercial kits are also available. Serum co-agglutination tests show a sensitivity of c. 90% in diagnosing pneumonia, while cerebrospinal fluid tests for meningitis show a sensitivity of c. 71–97% [10,11]. The performance of latex agglutination methods varies considerably, depending upon the clinical specimen. Latex agglutination had low sensitivity in detecting pneumococcal antigen in serum (18–47%) and urine (14–29%) [7], but was found to be very sensitive when testing sputum (90–94%) and blood cultures (100%) [12,13]. At the same time, latex agglutination is characterised by high specificity. The technique is a frequently performed laboratory procedure, but its clinical utility is controversial. A review of 5169 assays (for S. pneumoniae, group B streptococcus, Haemophilus influenzae and Neisseria meningitidis) performed over a 10-month period at two sites showed that only 57 (1.1%) of tests were positive, of which 31 (54%) were false-positives [14]. Therapy was not altered on the basis of any of the true-positive results, but false-positive results led to additional costs, prolonged hospitalisation and some clinical complications. Thus, this retrospective study did not support the use of latex agglutination for rapid antigen detection.
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Elisa ELISA-based rapid tests allow a diagnosis of pneumococcal pneumonia to be made with a sensitivity of >70% by the detection of capsular antigen in serum [15] or pleural fluid [16]. However, this method is not widely used and is not yet commercially available. Immunochromatography Immunochromatography is a modern and frequently used technique that can be used to determine the aetiology of pneumonia, meningitis and otitis media, to establish the diagnosis of invasive pneumococcal disease, and for the documentation of nasopharyngeal colonisation with S. pneumoniae. Several commercial kits based on immunochromatography are currently available. This technique is suitable for the identification of pneumococci in different types of clinical material, as well as for the detection of antigenuria in patients with pneumococcal diseases. Urinary antigen tests are 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 bacteraemic cases, the pneumococcal urinary antigen detected by immunochromatographic assay was still positive in 83% of cases after 3 days of therapy [17]. This form of urinary antigen testing has the principal additional advantages of rapidity (c. 15 min), simplicity and reasonable specificity in adults. Studies in adults have shown a sensitivity of 50–80% and specificity exceeding 90% [18,19]. The immunochromatography assay is also highly accurate in diagnosing pneumococcal meningitis (95% sensitivity with cerebrospinal fluid, 57% sensitivity with urine, and 100% specificity) [20]. The disadvantages of urinary antigen testing include the cost and the lack of an isolated organism for in-vitro susceptibility tests. Notably, immunochromatography is not suitable for evaluation of therapeutic effect, because positive test results are obtained for several weeks after recovery. Moreover, the immunochromatographic assays are non-specific for pneumococcal infections in children, particularly the very young, since nasopharyngeal carriage of S. pneumoniae can cause false-positive results [21]. Likewise, the
test may not be useful for predicting the aetiology of disease in populations with a high rate of pneumococcal nasopharyngeal carriage. Falsepositive results have been seen in patients with a prior episode of CAP within the previous 3 months, but they do not appear to be a significant problem in colonised patients with chronic obstructive pulmonary disease [18]. Recent studies have evaluated the clinical utility of urinary antigen testing for S. pneumoniae in patients with CAP. Guchev et al. [22] prospectively assigned patients with mild pneumonia to two groups. Those with positive urinary antigen test results were treated using pneumococcaldirected therapy with amoxycillin. Those with negative urinary antigen test results were treated with clarithromycin, based on perceived likelihood of infection by atypical pathogens. Of 219 evaluable patients, 22% had a positive urinary antigen test result. There was no difference in the outcomes of the two groups. Notably, 47 ⁄ 71 (62%) of patients in whom an atypical pathogen was identified were in the urinary antigen-negative arm, while 24 (38%) were in the urinary antigenpositive arm, indicating that they had S. pneumoniae in association with an atypical pathogen. However, since the atypical pathogens were determined by serological methods, these latter cases might have represented a primary atypical infection followed by secondary infection by S. pneumoniae. In such cases, it is probable that the clinical manifestations of infection that were treated were attributable to S. pneumoniae, and this may explain the good response to amoxycillin alone. The authors concluded that the urinary antigen test allowed them to administer targeted therapy with a penicillin-class antibiotic rather than a broader-spectrum agent. Such narrow-spectrum therapy can be more cost-effective and can allow broad-spectrum agents, such as macrolides or fluoroquinolones, to be reserved for patients whose urinary antigen test result is negative. Potential cost reductions are likely to be influenced by price differences between the targeted and broad-spectrum agents and by the proportion of positive test results [23]. Also, it should be noted that at the time of this study, in Russia, there was little b-lactam or macrolide resistance in S. pneumoniae. Thus, this approach may not be as useful in areas where high-level penicillin resistance exists. In addition, the study was performed
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in military trainees who were young (mean age, 19 years) and generally healthy. The authors suggested that additional trials are necessary for other clinical settings [22]. In another study, Stralin and Holmberg evaluated the urinary antigen test in 215 hospitalised patients with CAP, all of whom received initial b-lactam monotherapy [24]. The median age was 74 years, and approximately 45% of patients had a pneumonia severity index of class IV or V. Thus, these patients were more severely ill than those in the previously described study [22]. Thirty-eight patients had a positive urinary antigen result for S. pneumoniae, and 92% of these had a successful outcome; 114 had a negative urinary antigen result, and 78% of these had a successful outcome. There were no patients with a positive PCR sputum test result for an atypical pathogen in the urinary antigen-positive group, whereas six patients had a positive PCR result for Mycoplasma spp. or Chlamydophila spp. in the urinary antigennegative group. The authors suggested that a positive urinary antigen test result supports treatment with narrow-spectrum b-lactam antibiotics and that additional coverage for atypical pathogens is needed more frequently in patients with negative test results. MOLECULAR ASSAYS Molecular assays for S. pneumoniae include nucleic acid amplification techniques, i.e., PCR and loop-mediated isothermal amplification, and DNA hybridisation. PCR is a potentially attractive diagnostic tool for rapid diagnosis, since it does not rely on bacterial growth or the viability of the organism. There is some evidence that PCR-based assays are more sensitive than culture. PCR assays show moderate sensitivity (43%) in tests of blood, and high specificity in blood-culture-confirmed pneumococcal infections, and sometimes give positive results in culture-negative samples [25]. PCR techniques based on amplification of the pneumolysin or autolysin genes are applicable for the diagnosis of pneumonia, otitis media and meningitis. In sputum tests, autolysin and pneumolysin PCR has shown a high sensitivity (more than 80%), but a low specificity (30–40%) [26]. However, in pleural fluid PCR detects the pneumolysin gene with a sensitivity of 78% and a specificity of 93% [27].
The interpretation of sputum PCR tests is complicated by the difficulty in differentiating between pneumococcal colonisation and true infection. One approach that may help to resolve this problem is the quantification of the target organism by real-time PCR. Yang et al. evaluated the utility of a real-time pneumolysin gene PCR test using sputum samples from patients admitted with CAP [28]. Of 129 patients, 23% had S. pneumoniae isolated from blood or sputum. The sensitivity and specificity using real-time PCR were 90% and 80%, respectively. Real-time PCR can also detect S. pneumoniae in middle ear fluid from children with acute otitis media with a sensitivity of 100% and a specificity of 73% compared to culture [29]. Multiplex PCR allows the identification of several pathogens simultaneously in samples from patients with pneumonia, meningitis and otitis media with high levels of accuracy [30]. Loop-mediated isothermal amplification is a novel nucleic acid amplification technique that amplifies DNA under isothermal conditions (63C) with high specificity, efficiency and rapidity. It is characterised by a sensitivity 1000 times greater than that of conventional PCR [31]. PCR may therefore represent a useful diagnostic adjunct to assist clinicians in the selection of the most appropriate antibiotics. At present, conventional culture methods will still be required to confirm the antimicrobial susceptibility of the organism. However, PCR techniques also have the potential to be used for the rapid detection of antimicrobial resistance in S. pneumoniae. Amplification of the pneumococcal penicillin-binding protein 2B gene can be used to determine penicillin susceptibility, and provides susceptibility data even in culture-negative cases. The method has positive and negative predictive values of 100% and 91%, respectively, and showed 98.3% agreement with MIC data [32]. DISCUSSION The utility of diagnostic studies to determine the aetiology of CAP is controversial because of the lack of rapid, easily performed, accurate, costeffective methods that provide immediate results for the majority of patients at the point of service (i.e., the initial evaluation by a clinician in an office or acute-care setting). Nevertheless, there is a good rationale for establishing an aetiological
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diagnosis. The primary reason is to provide results that influence the antibiotic management of an individual patient. The selection of antibiotic therapy can be broadened, narrowed or completely changed on the basis of accurate diagnostic testing. Furthermore, management of early antibiotic failure can be greatly facilitated by the determination of an aetiological diagnosis. De-escalation or narrowing of antibiotic therapy on the basis of diagnostic testing may decrease costs, drug side-effects and the selection pressure for antibiotic resistance. These considerations are important in light of the specific impact of the rapid diagnosis of S. pneumoniae. Since all recommended antimicrobial options for empirical therapy in most of the published guidelines are effective against S. pneumoniae (including drug-resistant strains), it can be argued that there is little need to identify S. pneumoniae in order to ensure appropriate therapy for this pathogen. In addition, the possibility of polymicrobial CAP and the potential benefit of combination therapy for bacteraemic pneumococcal pneumonia have complicated the decision to narrow antibiotic therapy, since the identification of S. pneumoniae may not preclude a second pathogen. Furthermore, one randomised clinical trial of a diagnostic strategy in CAP demonstrated that, while there was a significant increase in diagnostic yield, there were no statistically significant differences in mortality or length of stay between pathogen-directed therapy and empirical therapy [33]. However, this study did not require the alteration or specification of therapy on the basis of the results of the diagnostic tests. Moreover, the study was performed in a country with a low incidence of antibiotic resistance (The Netherlands), which may limit its applicability to areas with higher levels of resistance. It is of interest that pathogen-directed therapy was associated in this study with a lower mortality rate in the small number of patients admitted to the intensive care unit. Adverse effects of treatment were significantly more frequent in the empirical therapy group, but may be unique to the specific antibiotic used (erythromycin). The absence of benefit overall in this study should not be interpreted as an absence of benefit for an individual patient. The identification of S. pneumoniae can be helpful in de-escalating antimicrobial therapy, a matter of particular importance in this era of antimicrobial resistance. Hopefully, a reduction
in antimicrobial use will lead to reduced pressure for emergence of resistance, as well as a reduction in adverse events. Knowledge of the causative pathogen can also contribute to the rational selection of antimicrobial agents when changing from intravenous to oral therapy in the hospital setting. Finally, regarding the issue of combination therapy for bacteraemic pneumococcal pneumonia, it is important to note that these studies evaluated the effects of initial empirical therapy before the results of blood cultures were known and did not examine the effects of pathogenspecific therapy after the results of blood cultures were available [34]. The benefit of combination therapy was also most pronounced in the more severely ill patients. Therefore, no data suggest that discontinuation of combination therapy, once results of cultures are known, is unsafe in non-intensive-care-unit patients. Thus, if an appropriate culture reveals the presence of penicillin-susceptible S. pneumoniae, a pathogenspecific, narrow-spectrum agent (e.g., penicillin or amoxycillin) can be selected, with the aforementioned potential benefits with respect to the selective pressure for resistance. CONCLUSION The rapid detection of S. pneumoniae provides a unique opportunity for more targeted use of narrow-spectrum antibiotics, thus creating the possibility of more rational antibiotic use. Further studies on the clinical utility of existing and newer technologies should provide the evidence required for the integration of rapid tests and early pathogen-directed therapy into routine clinical practice. The rapid diagnosis of S. pneumoniae for CAP will require a different approach to clinical management than is presently followed in many countries. Presently, the general standard of care for CAP is empirical therapy, with little emphasis on microbiological identification. However, we may be entering an era in which aetiological diagnosis will become paramount [35]. This is stated in reference to the identification of pathogens other than S. pneumoniae (e.g., communityonset methicillin-resistant Staphylococcus aureus and H5N1 Influenza A virus), which are not effectively treated by most recommended empirical antimicrobial regimens.
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