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agents in CAP management is notably simplified if the pathogen is defined.1,2

Additionally, given that the most of the forms of CAP are treatable, a better understanding of the causative pathogens could help outline efforts to define their natural history and optimize treatment.2

Common Etiologies of Community-acquired Pneumonia

Microbial etiology of CAP is not yet well-characterized.3Each case of CAP shows a variation in the causative agent/s implicated in its pathogenesis, which can be attributed to the regional differences in the prevalence of microorganisms. Nevertheless, there continue to be several common etiologies that lead to CAP both in the hospital and community settings.4

The ro le o f v i ruses in adul t CAP is not well-understood. They have been previously encountered in approximate ly 10% of adul t patients admitted to hospital with CAP. However, studies based on molecular diagnostics indicate an underestimation of the viral etiology in adult CAP owing to a previously limited range of diagnostic methods. The incidence of mixed infections involving viruses and respiratory pathogens is suggested to be common among patients hospitalized for CAP.3

The common pathogens implicated to cause pneumonia in ambulatory patients are Streptococcus pneumoniae (S. pneumoniae), Mycoplasma pneumoniae ( M . p n e u m o n i a e ) , H a e m o p h i l u s influenzae (H. influenzae), Chlamydia pneumoniae (C. pneumoniae) , and respiratory viruses, such as influenza A and B, adenovirus, respiratory syncytial virus, and parainfluenza. The common pathogens causing

Community-Acquired Pneumonia: Bacteriological Profile and Microbiological InvestigationsRavi Kumar1, Canna Ghia2, Shaumil Waghela3, Harish Thanusubramanian4, Gautam Rambhad5

pneumonia in hospi ta l set t ings include S. pneumoniae, M. pneumoniae, H. influenzae, C. pneumoniae, Legionella spp., and aspiration respiratory viruses, such as influenza A and B, adenovirus, respiratory syncytial virus, and parainfluenza. Severe pneumonia is caused by the pathogens S. pneumoniae, Staphylococcus aureus (S. aureus), Legionella spp., Gram-negative bacilli, and H. influenzae.4Atypical bacteria are predominant in patients with milder episodes of pneumonia and cases of ambulatory CAP.2

Streptococcus pneumoniae causes an est imated 1.6 mil l ion deaths worldwide. It is the most common pathogen caus ing CAP and an important reason for morbidity and mortality in the elderly, followed by S. aureus and Pseudomonas aeruginosa ( P . a e r u g i n o s a ) . 5 , 6 S t r e p t o c o c c u s pneumoniae also happens to be the most common etiologic agent isolated in CAP patients with severe disease and those requiring hospitalization. According to the population-based studies, S. pneumoniae was also the organism frequently isolated in outpatients with evidence of a similar etiologic profile across all age groups (both inpatients and outpatients).2

A detailed understanding of the pneumococca l ep idemiology i s essential, as the available vaccines target the polysaccharide capsule or the serotype-specific capsular protein.5

New and Emerging Etiologies of Community-acquired Pneumonia

In addition to the above mentioned common pathogens causing CAP, few studies undertaken have highlighted on the new and emerging etiologies o f CAP tha t can in f luence the management of the condition.4

1Prof Emeritus- Dept. of Microbiology, Principal Investigator- PIDOPS STUDY, Chief- Central Research Laboratory, KIMS Hospital and Research Centre, Bangalore, Karnataka; 2Senior Medical Advisor, 3Medical Advisor, 4 Area Medical Advisor, 5India Vaccines Medical Lead, Pfizer Limited, Mumbai, Maharashtra

Introduction

Lower respiratory tract infections (LRTIs) are a leading cause of

death due to infectious diseases worldwide.1 Of the LRTIs, community-acquired pneumonia (CAP) is the most important infectious disease of the respiratory tract encountered in clinical practice. It has an estimated incidence ranging from 1.6/1,000 to 16/1,000 per year.1,2

Diagnostic testing at one point of time was critical to antimicrobial agent selection, clinical outcomes, and per formance measures for management of the patient. However, over the past couple of decades, there has been an extraordinary decline in interest and supposed need for microbiological analysis in CAP cases. Also, the advent of antimicrobial agents to treat CAP has led to a progressive decrease in the perceived requirement to know the causative agent for which the patient is being treated.1

The microbiology of CAP may have probably remained stable over the past decade. However, there remains a possibility of change over time owing to an aging population and the success of vaccinations.2 The vast number of CAP studies from the pre-penicillin era showed that an etiologic diagnosis was established in > 90% of cases. Contrary to this, the 2009 data from Medicare indicate that a probable pathogen is now detected in < 10% based on a review of the records of > 17,000 patients hospitalized with CAP. The desire for better data-directed treatment toward pathogens clashes with harsh realities of cost, requirements for antibiotics to be given within 6 hours of disease onset, guidelines discouraging microbiological studies in most cases, and beliefs in empirical treatment approaches among others.1

While the initial antibiotic selection for CAP treatment remains empirical, consensus exists on the fact that selection of appropriate antimicrobial

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A prospective, multicenter study carried out by Fang et al . in 359 cases found S. pneumoniae (15.3%), H. influenzae (10.9%), Legionella spp. (6.7%), and C. pneumoniae (6.1%) to be the common causative pathogens of CAP. The highest mortality was seen with S. aureus (50%) and the lowest with C. pneumoniae (4.5%). The study highlighted on C. pneumoniae and Legionella spp. as possible emerging etiologies for CAP and the need for the observation to influence empiric antibiotic prescriptions.4

A new diagnostic platform using polymerase chain reaction (PCR) along with conventional methods evaluated the microbiological yield with an aim to identify emerging etiologies for CAP in a 12-month prospective study. Conventional testing included the culture of blood, sputum, and nasopharyngeal secretions. Other tests included analyses of sputum samples using real-time, quantitative PCR for S. pneumoniae, H. influenzae, and Moraxella catarrhalis [M. catarrhalis; nasopharyngeal secretion analysis using PCR; serological testing for M. pneumoniae, C. pneumoniae, and respiratory viruses; and the detection of pneumococcal and Legionel la pneumophila (L. pneumophila)] antigens using urine antigen assays.4

Adoption of the above mentioned approach helped to establish microbial etiology in about 67% of patients and identify a microbiological agent in 89% of them. The most frequently detected pathogens were S. pneumoniae (38%) and respiratory virus (29%). About

35% of patients were identified to be infected with two or more pathogens.4

Data from a recent case series have highlighted on methicillin-resistant S. aureus to be a cause for severe CAP. However, it remains an uncommon cause, and the prevalence and risk factors are not known.7

Bacteriological Profile of Community-acquired Pneumonia

The bacteriological profile of CAP is seen to vary across countries and within the regions of the same country. This fact is attributed to factors like environmental pollution, differences in frequency of use of antibiotics, awareness of the disease, and life expectancy. However, the most common pathogen across countries, such as India, Europe, the United Kingdom, and the United States, is S. pneumoniae.4

Indian scenario

Etiologic agents of CAP vary across the different geographical areas of the country. Streptococcus pneumoniae predominates as the etiologic agent in Shimla and Delhi. Other frequently isolated pathogens identified in about 75% of patients from Shimla were S. aureus, Klebsiella pneumoniae (K . pneumoniae ) , M. pneumoniae, Escherichia coli (E. coli), and other Gram-negative bacteria. The organisms isolated from CAP patients in a prospective study conducted in Mumbai included S. pneumoniae, C. pneumoniae, H. influenzae, M. catarrhalis,

M. pneumoniae , L. pneumoph i l a , P. aeruginosa , Staphylococcus , and S a l m o n e l l a t y p h i . S t r e p t o c o c c u s pneumoniae was the leading cause of CAP, isolated in 22% of patients; while atypical organisms and Mycobacterium tuberculosis were identified in 19% and 7% of patients, respectively.4

In a study among hospitalized patients, S. aureus was the primary causative organism identified. Other pathogens identified included isolates of Pseudomonas and Klebsiella . In critically ill patients, most likely isolates include Enterobacteriaceae, S. aureus, and P. aeruginosa.4 In yet another study recently conducted in 2015 among CAP patients in Mumbai, the common organisms isolated from sputum culture were as given in Figure 1. Streptococcus pneumoniae, Pseudomonas, E. coli and Acinetobacter, S. aureus, Klebsiella, and mixed infections were found in 56%, 11.1%, 5%, 3.3%, 1.3%, and 16.7% of patients, respectively.8

Tests to Determine Etiology of Community-acquired Pneumonia

The knowledge of pathogens that cause CAP is the basis for the selection of empirical antibiotic treatment that in turn has a substantial impact on the patient prognosis. However, the establishment of a microbial diagnosis for patients with CAP continues to pose a challenge.3 The current guidelines on CAP discourage the approach based on microbiological diagnosis for pneumonia in non-hospitalized patients, which is probably due to lacking sensitive, specific, and cost-effective diagnostics. Although retrospect ive s tudies may have revealed favorable outcomes using empiric therapy in CAP patients, failing to accurately identify the causative agent as S. pneumoniae has far-reaching consequences both for the affected individual and the population as a whole. At the patient level, it may result in an inadequate antimicrobial therapy or overly broad-spectrum and expensive treatment, t r a n s l a t i n g i n t o l i m i t a t i o n s i n assessing disease burden, evaluating the effects of immunization, tracking of antimicrobial resistance, and exploring new therapeutic agents among the population.5

Traditionally, the diagnosis of CAP has been made using the conventional method of culture of respiratory

Fig. 1: Most common organisms isolated in CAP patients

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secretions (sputum, bronchoalveolar lavage, and pleural f luid) or by detecting pneumococcal bacteremia. Yields of these, however, are low.5

Serological tests may be performed additionally.4 Limitations exist with the use of these methods given that the sensitivity of blood culture is around 10–15%, while that of sputum culture is between 10 and 30%. Even with the use of invasive sampling techniques, such as bronchoalveolar lavage or transthoracic aspiration, etiology of CAP remains undetected in 40% of cases.9

Culture-based methods

The advantages of these methods include the ability of implementation worldwide with low cost and high specificity and the capability to provide data on antibiotic susceptibility and serotypes.5 Potential specimens for Gram staining and culture in CAP may include blood, sputum, pleural fluid, or lung aspirate.10

Blood and/or pleural fluid culture

Blood samples for culture are easy to obtain and can provide a definite microbial diagnosis when the results are positive.3 However, blood cultures are rarely positive. If detected posit ive, i t may indicate severe disease.4 Moreover, CAP caused by typical bacterial pathogens, such as S. pneumoniae and H. influenzae, is frequently associated with positive blood cultures. Bacteremia with M. catarrhalis is rare.11 The utility of blood and pleural culture in the etiologic diagnosis of the condition is, however, hampered by the limited sensitivity of these diagnostic approaches.6

Sputum culture

Expectorated sputum is the most readily available specimen and the diagnostic standard in the majority

of cases. However, microbiological samples, such as sputum, are subject to oropharyngeal contamination, and hence, pose difficulty in detecting relatively fastidious bacteria like S. pneumoniae. While some laboratories continue to perform tradit ional methods of Gram staining and culture or Quellung staining to distinguish S. pneumoniae , these approaches have become relatively unusual.1 The sensitivity and specificity of sputum Gram stain vary among the different settings. Compared to culture, the sensitivity of this test ranges between 10% and 15%, while its specificity ranges from 11% to 100%.6 Gram staining and culture to identify S. pneumoniae is problematic as a result of false-positive and false-negative results.1

As against the pneumococci, S. aureus and Gram-negative bacteria are easily detected using selective media. Although rare, these organisms are likely to be disproportionately represented, especially in specimens o b t a i n e d p o s t - t r e a t m e n t w i t h antibiotics.1

A s p u t u m c u l t u r e c a n n o t d i f f e r e n t i a t e p a t h o g e n f r o m commensal and is of no value if obtained after starting antibiotics, as it will only lead to the detection of colonizers. Identification of microbe is possible only in 50% of patients and depends on the quality of sputum sample.4

Urinary antigen test

Detecting pneumococcal antigen in urine is the most widely used indirect method for S. Pneumoniae detection of pneumococcal antigen in urine. The group C polysaccharide cel l wall antigen is detected by this test. It is useful in diagnosing

pneumococcal pneumonia.5 It is a quick, sensitive, and specific test to detect pneumococcal CAP in adults and it may remain positive for many weeks after pneumococcal pneumonia.12

The advantages inc lude ease of getting a diagnostic specimen, greater diagnostic yield compared to sputum, and ability to establish the diagnosis even after antibiotic treatment init iation. The test as eva luated in s tudies showed a sensitivity of 82% and a specificity of 97%. The sensitivity and specificity are, however, less in adults with non bacteremia pneumonia.1 The main disadvantage noted with this test is its false-positive results in patients with nasopharyngeal colonization and those with recent episodes of pneumococcal infections.6 However, it is the favored method for the detection of Legionnaires disease.1

The assay has a significant role to play in the rapid etiologic diagnosis of CAP, particularly in patients unable to produce good quality sputum, who are not bacteremic, and in whom antibiotics have been administered before admission.6 It significantly increases the diagnosis of a pneumococcal origin beyond standard microbiological cultures. It raises the intriguing possibility that considerably more cases of CAP are caused by S. pneumoniae than that conventional tests can currently confirm (Figure 2).9

Molecular diagnostics

The traditional microbiological m e t h o d s f o r t h e d e t e c t i o n o f respiratory tract pathogens can be slow. They are often not sensitive, fail to differentiate infection from colonization, and are influenced by previous antibiotic therapy. Molecular diagnostics are more promising in the detection of the common and atypical bacterial pathogens that can cause CAP. Molecular analysis can detect typical pathogens in hours and atypical pathogens in weeks. This approach aids to eliminate concerns about decreased viability of the organism associated with the effects of previous antibiotic therapy and transport of specimens.13

Current molecular diagnostic methods have a great potential to include targets useful in the rapid identification of microorganisms and antimicrobial resistance. They aid to analyze directly unprocessed

Fig. 2: Improved rate of detection of causative agent in CAP

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samples and to obtain quantitative results in pneumonia. Molecular methods compared to traditional methods have superior sensitivity, relatively rapid turnaround time, and are able to identify pathogens that are slow growing or difficult to culture. A variety of respiratory samples including expectorated sputum, bronchoalveolar lavages, protected bronchial brushes, and endotracheal aspirates are amenable to molecular testing. These assays may target either a single pathogen or multiple respiratory pathogens in a single assay.14

Nucleic acid detection with the use of PCR has the advantage of being able to identify pathogens in patients already on treatment with antibiotics. Molecular and antigen-based testing possess the advantage of being able to deliver rapid results before initiation of treatment.5 Furthermore, PCR is useful to identify atypical bacterial strains, including Mycoplasma, C. pneumoniae, and H. influenzae type b. It is more rapid as compared to virus isolation and serology and is also more sensitive. Of recent, the FDA has cleared the PCR assay for the detection of 12 respiratory tract viruses. Rapid diagnostic tests for detecting influenza viruses show good specificity but a sensitivity of only 50‒70%.15

Conventional PCR, real-time PCR, and in-house or commercial multiplex mPCR are considered to be more specific than cultures and serology. A mPCR method compared the diagnostic yields that were obtained f rom mPCR of nasopharyngeal aspirates, nasopharyngeal swabs, and induced sputum ( IS ) wi th those obtained with specific PCR commercial kits, paired serology, and urinary antigen. All the PCRs were seen to have good specificity but low sensitivity in nasopharyngeal samples. The IS sample had the best performance for the diagnosis of M. pneumoniae by PCR. Sensitivity and specificity of upto 100% was stated to be obtainable if the characteristics of the population, the type of respiratory sample, and the inhibitors present in each of them is known. These tests, however, did not allow for discrimination between patients with acute or convalescent infection and asymptomatic carriers.16

Implementation of comprehensive molecular testing of single lower respiratory tract specimens achieved

pathogen detection in 87% of patients (adults hospitalized with CAP) as compared to 39% with culture-based methods. There was significantly greater detection of H.influenzae, S. pneumoniae, M. catarrhalis, S. aureus, E. coli, and K. pneumoniae than standard culture-based methods. Polymerase chain reaction significantly detected bacteria in 143 culture-negative specimens. In addition a higher bacterial load was detected in culture-positive specimens as compared to culture-negative specimens. It also provided valuable information about individual bacterial loads. It was seen to significantly improve pathogen detection in CAP, especially in patients exposed to antimicrobials and enabled early de-escalation from broad-spectrum empir ical antimicrobials to pathogen-directed therapy. Reporting of results was done in a clinically relevant time-frame.17

In yet another study two diagnostic bundles that included sputum and b lood cul ture , ur inary ant igen detection, and nasal swabs for PCR probes and at least one measurement of procalcitonin level were compared in patients (127 in number ) with CAP. While an etiologic diagnosis was established in 71% of patients, a respiratory virus was detected in 39%. The potential of molecular diagnostics and serum procalcitonin (PCT) levels for improved antibiotic stewardship was evident in 25 patients with only detectable respiratory virus and normal levels of PCT.18

Some of the contr ibut ions of these novel molecular diagnostic approaches include multiplex assays, user-friendly formats, providing of results in a few hours, high sensitivity and specificity in identification of pathogens, detection of antibiotic r e s i s t a n c e g e n e s , a n d t a r g e t quantification. Future challenges may include the development of new molecular tests for other bacterial respiratory pathogens, detection of pathogens and new key antimicrobial resistance genes in unprocessed samples, and determination of the microbial load by quantitative multi-pathogen tests.14

Serologic tests for community-acquired pneumonia

The immunoglobulin M (IgM) antibodies against Mycoplasma are seen to rise one week after infection and persist to be positive for 6 months

to 1 year after infection, resulting in poor sensitivity and specificity of the test. It takes 3 weeks for IgM antibodies to Chlamydia to become detectable, r e s u l t i n g i n p o o r s e n s i t i v i t y . Mycoplasma can be diagnosed by cold agglutinins (sensitivity of 50% and poor specificity) and elevated reticulocyte count. The limitations are that it requires rising titers, is expensive, and is not readily available.4

Antibody tests

Diagnost ic tests may include the detection of antibodies to S. pneumoniae, Mycoplasma, Chlamydia, adenovirus, influenza viruses (A and B), parainfluenza viruses (1, 2 and 3), respiratory syncytial virus, etc.15

Spectrum of Pneumococcal Disease

Pneumococcal disease may be classified as invasive or non-invasive disease. Pneumonia, otitis media, and sinusitis are the classical presentations of non-invasive diseases. Invasive disease is characterized by sepsis, bacteremic pneumonia, meningitis, a n d a r t h r i t i s . 4 S t r e p t o c o c c u s pneumoniae, a Gram-positive, catalase-negative organism, is responsible for pneumococcal infections. It causes a wide spectrum of disease pathologies that include CAP, bacterial meningitis, bacteremia, otitis media, sinusitis, s e p t i c a r t h r i t i s , o s t e o m ye l i t i s , peritonitis, and endocarditis owing to its ability to directly extend from the nasopharynx into adjacent anatomic structures or vascular invasion with a hematogenous spread.10

Pneumonia is the most common invasive pneumococcal disease, S. pneumoniae being the most frequent cause of bacterial pneumonia due to a known pathogen. I t is also suggested to be the most common pathogen in culture-negative disease. However, the actual proportion of CAP caused by S. pneumoniae is difficult to determine given the lack of a gold standard for diagnosis.5 Up to 60% of individuals in the community have nasopharyngeal carriage of S. pneumoniae.4

Difficulty in Diagnosis and Identification of Streptococcus pneumoniae

Streptococcus pneumoniae is an important human pathogen having

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the potential to cause a broad range of diseases. However, microbiologic confirmation of the pathogen can be difficult.5 While S. pneumoniae has been the most common identifiable pathogen in CAP, there has been a remarkable reduction in the frequency of detection of the pathogen since the 96% diagnostic yield achieved in the pre-antibiotic era, with more recent reports indicating a yield of only 10–20%.1

R e g a r d l e s s o f t h e p o t e n t i a l benefits of the diagnostic tests for S. pneumoniae, issues pertaining to both sensitivity and specificity for disease, depending on the clinical setting and type of specimen tested, exist. Interpretation of diagnostic testing for S. pneumoniae needs to address the fundamental questions of whether the test in question specifically identifies the pathogen and if the detection adequately impl icates i t as the causative pathogen of the disease.5

Although the most common cause of CAP, it remains undoubtedly underdiagnosed. Isolat ion from blood is specific but lacks sensitivity, while the isolation from sputum may represent colonization.9

S t r e p t o c o c c u s p n e u m o n i a e i s identified from the culture by accurate observat ion of i t s morphologic a p p e a r a n c e a n d p h e n o t y p i c characteristics of α-hemolysis of blood agar, optochin susceptibility, catalase negativity, and bile solubility. Although the phenotypic markers are quite reliable, the finding of optochin-resistant pneumococci has decreased the use of this characteristic. Streptococcus pneumoniae is a lso difficult to obtain in a culture given the tendency of the pathogen to autolyze on reaching the stationary phase of growth, in patients in whom antibiotic treatment has been initiated before collection of specimen, and l imitations such as diff iculty in adequate collection of specimen and low prevalence of detectable b a c t e r e m i a i n C A P . T h e f i r s t commercialized assays were the tests based on the capsular polysaccharide antigens of S. pneumoniae. But these have poor sensitivity and specificity

as compared to standard Gram stain and culture.5

While empiric therapy for CAP is directed at S. pneumoniae, there may be an involvement of other pathogens, such as atypical bacterial pathogens or respiratory viruses. An accurate and reliable finding of S. pneumoniae would benefit both pneumococcal and non-pneumococcal disease.5

Summary

The role of microbiological testing for CAP continues to be debatable. Even with the best available diagnostic methods, a specific etiologic agent is detected in only 50% of CAP cases. The conventional laboratory tests for pathogens causing CAP are so poor that even current clinical practices recommend testing for only the severely affected individuals.13 It is essential to determine the cause of CAP, whether it is caused by a bacterium, atypical bacterium, or virus, as they all need different treatment approaches.15 The need for identification of S. pneumoniae is essential, as pneumococcal pneumonia is considered to be the main etiology due to cultivable bacteria.9

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16. Herrera M, Aguilar YA, Rueda ZV, Muskus C, Velez LA. Comparison of serological methods with PCR-based methods for the diagnosis of community-acquired pneumonia caused by atypical bacteria. J Negat Results Biomed 2016; 15:3.

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