Determination of pneumonia etiology is ideally based upon isolation of causative pathogens, or characteristic histology in the case of non-infectious pneumonia, at the site of disease (23). However, effective sampling from the lower respiratory tract (LRT) is frequently not clinically feasible. Upper respiratory tract (URT) specimens such as nasopharyngeal (NP)/oropharyngeal (OP) swabs must often serve as a proxy. These URT specimens can be particularly difficult to interpret due to confounding by asymptomatic carriage of non-pathogenic organisms (24).

The nasopharynx is a complex and dynamic environment; any organism that could cause pneumonia may also be a colonizer, including Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis (17, 25, 26). Furthermore, asymptomatic NP carriage may responsible for transmission and often precedes LRT infection (27). Klebsiella pneumoniae is an opportunistic gram-negative bacteria that can colonize the NP, although it more commonly colonizes the gut (28, 29). Studies have reported a lower prevalence of NP carriage in children (range 1.4–7%) than in adults (20–32.5%), which may be related to poor hygiene (29–31). However, in terms of its pathogenicity, the presence of K. pneumonia in symptomatic children should be considered a pathogen (28, 32).

Previous studies have suggested that children with pneumonia have a higher pathogen density in the URT than children without pneumonia, though there is heterogeneity by study and pathogen (25, 33). Likewise, respiratory viruses found in respiratory specimens by molecular tests during acute disease may be “innocent bystanders” without a causal role (34). The presence of a respiratory virus, such as human Bocavirus (hBoV), Adenovirus (AdV), non-SARS human Coronavirus (hCoV), Enterovirus (EV), and Rhinovirus (RV), in the URT of healthy children has been observed in several studies (13, 19, 20). This could represent carriage, nascent infection, prolonged shedding, or subclinical infection (18, 34, 35). Some respiratory viruses, such as hBoV, can shed for several months after an illness (36).

Secretions from the LRT are also diagnostically helpful as they originate at the site of infection (24). However, children have difficulty expectorating sputum, leading to frequent use of induction techniques to obtain LRT specimens. Sputum induction is performed by administration of hypertonic saline via nebulizer, followed by percussion of the chest wall to mobilize secretions (21). The presence of <10 squamous epithelial cells (SECs) and significant number of polymorphonuclear cells/PMNs (for which criteria vary among studies) per low-power field (100× magnification) have long been regarded as ideal (24, 37). However, the criterion of large numbers of PMNs has been questioned given that pneumonia may not necessarily be associated with production of purulent sputum (38). Recent studies showed <10 SECs is the key quality measure in children since it corresponds with low quantities of NP/OP flora (38, 39). However, interpretation should take into account that contamination may occur even with meticulous technique (24).

Microbiologic analysis of LRT specimens, including induced sputum, by techniques such as culture and gram stain can reveal an etiology and guide targeted management (21, 40). Polymerase chain reaction (PCR) for bacterial DNA by is particularly helpful in culture-negative cases, highlighting the complementary role of molecular methods (41). Results should be interpreted in the context of immune status, as opportunistic pathogens such as Pneumocystis jirovecii and cytomegalovirus (CMV) are often pathogenic in children with HIV but not in HIV-uninfected children (42).

Etiologic assessment of pneumonia may also be done using blood samples, which may be easier to collect than LRT specimens and NP swabs. As blood is normally sterile, culture of an organism is generally considered indicative of active infection (24). However, prevalence of blood culture contamination may be as high as 2–3% (43) and bacteremia only occurs in 2.1% of pneumonia cases (44). Thus interpretation must account for the possibility of contamination, which we have incorporated in our pathogen identification rules. Blood specimens may also undergo molecular testing. This is especially helpful for some organisms. For instance, PCR detection of S. pneumoniae in blood may be associated with invasive pneumococcal disease (44, 45).

Antigen detection on several specimen types, including urine, pleural fluid and NP/OP swabs, is commonly used for point-of-care (PoC) diagnosis (23). Although antigen detection tests are only available for select organisms, they can play a critical role in treatment decisions. For example, rapid recognition of S. pneumonia or Legionella pneumophila, can guide antibiotic selection. A limitation of antigen detection assays is their reliance on detectable quantities of antigen; performance is thus suboptimal compared with molecular tests (45).

Serological testing of paired acute and convalescent samples, typically collected at least 7 days apart, can also be used for pathogen identification (23). Serology is most helpful for retrospective confirmation, especially for fastidious bacteria, and may reveal the accuracy of a previous empiric diagnosis (45). It may be particularly useful for viruses with prolonged NP shedding or high prevalence in a control population (24). IgM only tests are less sensitive and specific than IgG and IgM antibody titers from paired specimens as IgM kinetics vary (23) and IgM seroconversion might not occur in the setting of repeat infection (46). A serologic diagnosis generally requires a 2-fold or greater increase in titers between paired serum specimens. Reliance on convalescent specimens means that actionable results are not typically available during acute illness, limiting application in the acute-care setting (47).

Our rules to determine the bacterial etiology of pneumonia incorporate commonly collected specimens and routine laboratory assays. Blood culture and whole-blood PCR are considered first-tier due to high specificity, followed by induced sputum culture and molecular testing (PCR) of NP/OP swabs or induced sputum specimens (45). Serological evaluation of paired acute and convalescent specimens is also used for diagnosis, especially of atypical bacterial agents (48) for which PCR showed low sensitivity and poor concordance compared with the paired serology (49). The proposed pathogen determination rules are shown in Table 1.

All bacteria detected by blood culture, except for contaminants, are considered potential pathogens. The following were classified as contaminants based on relatedness to community-acquired pneumonia: Coagulase-negative staphylococci, Micrococcus spp., Propionibacterium spp., Alpha-hemolytic streptococci (except pneumococcus, Streptococcus anginosus, and Streptococcus mitis), Enterococcus spp., Corynebacterium spp. (diphtheroids), Bacillus spp. (except Bacillus anthracis), Pseudomonas spp. (except Pseudomonas aeruginosa), Stomatococcus, Aeroccocus, Neiserria subflava, Veillonella spp., other environmental non-fermenting Gram-negative rods, and Candida spp. (14, 20). Any bacteria found in whole blood by PCR and not listed as a contaminant is classified as a pathogen (20).

Good quality sputum specimens should undergo gram stain and bacterial culture simultaneously to optimize microbiological yield (21, 50). Gram stain results that are consistent with sputum culture can define the microbiological diagnosis of pneumonia (21). The gram stain should be interpreted as follows: Gram-positive lancet-shaped diplococci (GPDC) suggest S. pneumoniae; Gram-positive diplococci (GPDC) or cocci in chains suggest Streptococcus pyogenes; Gram-positive cocci in clusters (GPC-cluster) suggest S. aureus; Gram-negative coccobacilli (GNCB) suggest H. influenzae, Bordetella pertussis or Acinetobacter baumannii; Gram-negative diploccoci (GNDC) suggest M. catarrhalis; large Gram-negative rods (GNR-large) suggest Klebsiella pneumoniae or Escherichia coli; and small Gram-negative rods (GNR-small) suggest P. aeruginosa (40).

For semiquantitative measurement on sputum culture, predominant organisms were identified and quantified according to the furthest quadrant with visible colonies (first quadrant, scanty; second quadrant, 1+; third quadrant, 2+; fourth quadrant, 3+). Organisms isolated in quantities of 2+ or 3+ and with compatible Gram stain morphotype are regarded as pathogens (38). For quantitative culture, colony counts of <10⁴/ml suggest contamination, counts of 10⁴-10⁵/ml are indeterminate, and counts of >10⁵/ml of a major isolate suggest a potential pathogen (51). If the organism grows on culture, conventional biochemical tests (e.g., catalase or coagulase) or automated system evaluations, including rapid fluorescence-based methodology, can be performed to identify the organism (52, 53).

Culture yield depends on sputum quality, pathogen burden, transport time, storage period, nutrients, and incubation conditions that maintain viability (50, 54). Receipt of antibiotics before specimen collection may prevent growth on culture (41) as well as affect gram stain morphology. In this setting, culture-negative bacteria should remain in the differential and be sought by a molecular method targeting DNA (41). For bacteria identified by PCR of respiratory specimens (NP, OP or induced sputum), careful consideration must be given to distinguishing colonizers from true pathogens (14, 17).

For bacteria not considered NP colonizers (e.g., Mycoplasma pneumoniae), any positive PCR indicates a definite pathogen (13, 19). For bacteria than can be both a colonizer and pathogen, higher density has been associated with pathogenic status. For example, higher density is associated with causality and considered significant if the copy number exceeds 6.9 log₁₀ copies/mL for S. pneumoniae (26), 5.9 log₁₀ copies/mL for H. influenzae (25), and 7.5 log₁₀ copies/mL for S. aureus (33). Copy number cut-offs were transformed to corresponding Ct values per our assay; the Ct cut-off for S. pneumonia was ≤ 24.2; H. influenzae ≤ 30.3 and S. aureus ≤ 30.2. We nonetheless recognize that Ct value cut-offs may differ depending on the PCR system used. Lower copy numbers are considered indicative of colonization. However, studies have failed to identify an association between density and pathogen-confirmed pneumonia for M. catarrhalis, indicating that PCR alone cannot be used to determine its role in childhood pneumonia (25, 33).

Despite limited use in acute management of pneumonia, serological techniques are heavily utilized in clinical research (45). In comprehensive epidemiology studies, serology may identify infections missed by other methods such as culture and PCR (45) and could also distinguish colonization since nasopharyngeal carriage does not cause seroconversion between paired sera (55). A study in children over 2-years old throughout Asia demonstrated that a combination of paired serology, direct antigen and DNA tests increases diagnostic yield of atypical pathogens such as M. pneumoniae, Chlamydia pneumoniae, and Legionella pneumophila (56).

For interpretation of paired serologic testing, the following are considered indicative of infection: (a) Initial detection of specific antibodies after a prior negative sample (seroconversion), or (b) A two to four-fold increase, depending on the test, in antibody titers in the convalescent specimen (47). Serologic confirmation of pneumonia etiology is especially useful in prolonged hospitalization/ICU admission cases (57), and epidemiologic research or disease surveillance, as opposed to the acute clinical setting (45).

At present, few antigen detection assays are available for diagnosis of bacterial causes of childhood pneumonia. Notable examples include antigen detection assays for S. pneumoniae and L. pneumophila (45). The immunochromatographic urine test used for pneumococcal disease detects the polysaccharide cell wall antigen (58). Unfortunately, the test is not reliable in children since it cannot distinguish between carriage and pathogenic pneumococcus (59). Likewise, detection of soluble Legionella antigen in urine is limited to L. pneumophila serogroup 1 (60). Given their low sensitivity compared with PCR testing, use of these antigen assays in children is not recommended (45) and not included in our proposed rules.

Detection of viruses by direct immunofluorescence microscopy and isolation in cell culture have long been considered the “gold standard” of respiratory viral pathogen diagnosis (45). However, viral culture is resource-intensive, requires maintenance of cell-lines over long time periods, and poses exposure risk to laboratory personnel (61). Direct fluorescent-antibody (DFA) and immunofluorescent-antibody (IFA) assays of cell smears to detect specific viruses are commercially available and use standardized reagents (e.g., FLUA/B, PIVs 1 to 3, ADV, hMPV, and RSV), but are labor-intensive, require a fluorescence microscope and skilled microscopist, and are susceptible to reader error (62). Therefore, these “gold standard” methods are being replaced by more sensitive, high-throughput, and less labor-intensive molecular tests (45, 48).

A PCR test on NP/OP swabs or induced sputum with Ct (cycle threshold) value ≤ 40 is considered diagnostic for viruses well-known to cause pneumonia, including Respiratory syncytial virus (RSV), Influenza virus (IV), Human metapneumovirus (HMPV), and Human parainfluenza viruses (HPIVs) (13, 19). However, some viruses normally detected in healthy children or that have prolonged/intermittent shedding (innocent bystanders), including RV (63), AdV (64), hBoV (65), non-SARS hCoV (13), and EV (66), merit additional justification. To avoid attributing disease to innocent bystanders, we consider only those with high viral load (Ct value <24) as true pathogens (18, 67).

Viruses confirmed by serodiagnosis are also regarded as causative pathogens. Serology may be particularly useful for identifying respiratory viral infections that trigger secondary bacterial pneumonia as the virus may no longer be detectable in respiratory specimens (45). Addition of serology to PCR evaluation increases positive diagnostic yield (68). In one study, 29 of 88 cases of viral pneumonia were diagnosed by serology alone (69). Serology is a helpful adjunct to the PCR in assessing etiologic contribution, but may not reveal exact timing (68).

Antigen testing for respiratory specimens is attractive as a potential PoC diagnostic test for viral causes of childhood pneumonia. It has been used for detection of seasonal influenza and RSV (45). However, performance is suboptimal; sensitivity of rapid diagnostic tests ranges from 10 to 96% for influenza (70, 71) and 71–95% for commercial RSV (72, 73). Variable sensitivity is not surprising, as low viral loads would predispose to false negatives. Furthermore, low pre-test probability outside respiratory virus season undermines predictive utility (62). Antigen destruction may occur with freezing and on repository swabs, which can also decrease accuracy (74). Based on sensitivity, antigen testing is inferior to PCR for detection of viruses causing pneumonia (62) and is not included in our rules.