Principles of Testing

The decision to order a diagnostic test should hinge on whether the result is likely to affect the clinician’s treatment decisions. If the clinician is certain the patient has a disease based on clinical presentation and prevalence (high pretest probability), then the decision to treat will likely not be altered by the test result and testing should not be ordered. Similarly, testing should not be ordered if the clinician has a high degree of a priori certainty that the patient does not have a disease, because the decision not to treat will likely not be altered by the test result. Testing is most useful when the clinician is uncertain about the probability of disease and the result can sway the physician’s decision about treatment. In addition to the pretest probability, several factors affect this decision. For example, if therapy comes at a low harm (in terms of toxicity, dollar cost, and selection of resistance), then treating all patients without testing may be appropriate. If the diagnostic has a low sensitivity (i.e., the test is positive in a low percentage of patients with disease), then testing may lead to an inappropriate decision not to treat. Similarly, if a diagnostic has a low specificity (i.e., the test is positive in a high percentage of patients without disease), then testing may lead to unnecessary treatment. The determination that clinical suspicion is uncertain enough to benefit from a particular diagnostic involves the interplay of the cost and accuracy of the diagnostic test, the pretest probability of the disease, and the benefit and harm of treatment.

Infection Prevention

The clinician plays a critical role in notifying the micro­biology laboratory (and the hospital infection control epidemiologist) when virulent and transmissible agents are suspected as the cause of disease. Alerting laboratory staff reduces the exposure risk of laboratory staff handling specimens and cultures harboring highly virulent pathogens. A list of such pathogens is shown Table 17-1 . Not all specimens from patients with infectious diseases should be handled by the on-site laboratory. According to guidelines developed by local and national public health officials, specimens potentially containing selected high-risk agents such as Bacillus anthracis spores, Francisella tularensis, Yersinia pestis, variola major, hemorrhagic fever viruses, or Clostridium botulinum toxin are directly sent to the public health laboratories, where appropriate containment facilities and diagnostic tools are applied to make a diagnosis. Other pathogens that are handled by the on-site laboratory but still require laboratory notification include Coccidioides and Brucella species, because cultures of these are associated with a high risk for laboratory-associated infection. Although the technologists are expected to handle all specimens and microbiologic cultures using universal precautions, accidental exposures can happen, especially if the findings are unexpected. Therefore laboratory notification serves to alert the staff to protect themselves from potential exposure to highly transmissible agents.

Syndromic Order Sets

The diversity of etiologic agents of lower respiratory tract infection poses a number of diagnostic challenges to the clinician. First the provider must formulate a comprehensive yet pragmatic differential diagnosis that takes into account the clinical presentation, immune status, and the exposure history of the patient. Then the clinician must order the correct set of laboratory tests and ensure collection of the appropriate specimens and their placement in correct transport containers as well as their transport to the laboratory under permissive conditions for testing. Because improper test selection and specimen collection could reduce the analytic sensitivity and specificity of assays performed in the laboratory, syndromic order sets have been designed that consider the most common pathogens for the specific syndrome. Syndromic order sets incorporate general guidelines for the types of specimen required, collection and transport, and available assays for pathogens expected in a given clinical setting or syndrome. By prioritizing diagnostics that maximize yield and avoiding the need to repeat invasive procedures, these order sets also serve to minimize risk to the patient and to lower health care costs. However, it is the responsibility of the clinician to ensure that specimen requirements are met and the most critical tests are prioritized, especially when the amount of specimen material obtained is limited and multiple tests are ordered. Tables 17-2, 17-3, and 17-4 show syndromic order sets for community-acquired pneumonia (CAP), hospital-acquired and ventilator-associated pneumonia, and immunocompromised host pneumonia, respectively. Order sets developed to address local epidemiologic characteristics and preanalytic practices may be tailored to serve each institution. Clinicians also should familiarize themselves with local sample storage practices in case additional tests need to be performed.

Specimen Selection, Collection, and Transport

In general, sterile specimens such as tissue samples and aspirates are the most valuable diagnostically because the absence of contamination with commensal organisms ensures that any organism detected likely represents a true pathogen. Histopathologic examination of tissue also provides information on the immunopathologic characteristics of the infectious process. However, a major diagnostic challenge of lower respiratory tract infection is that lower respiratory tract secretions are usually obtained through the oropharynx, which normally contains 10 ¹⁰ to 10 ¹² colony-forming units (CFU) of aerobic and anaerobic bacteria per milliliter. Therefore lower respiratory tract secretions collected for microbiologic examination are commonly contaminated with diverse bacteria ( Table 17-5 ), some of which, such as Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Neisseria meningitidis, can also be pathogens of the lower respiratory tract. The oropharynx can also contain Mycoplasma pneumoniae and aerobic actinomycetes including Nocardia and nontuberculous mycobacteria in the absence of disease. In addition, aspiration of even minute amounts (0.1 to 1 µL) of oropharyngeal secretions can deliver a bolus of 10 ⁹ CFU to the tracheobronchial tree. The distinction in such cases between colonization of the upper respiratory tract and pneumonia cannot be easily made by sputum examination and culture. Another challenge is that oropharyngeal secretions, which normally contain only a few gram-negative bacilli (such as Enterobacteriaceae, Pseudomonas, Acinetobacter ), often become colonized with as many as 10 ⁷ CFU of gram-negative bacilli per milliliter in seriously ill patients requiring intensive care, patients treated with antibiotics after hospitalization for acute pulmonary inflammatory disease, chronic alcoholic and diabetic patients, institutionalized older adults and chronically ill patients, and hospitalized patients with acute leukemia. Lastly, Aspergillus spores present in the environment are commonly deposited in the lower respiratory tract and may be recovered from sputum in the absence of disease, although in immunocompromised patients it is best to consider this finding seriously. In summary, because lower respiratory tract secretions collected through the oropharynx are nearly always contaminated with resident microflora of the oral cavity and definitive diagnosis would require sterile lung tissue with demonstration of parenchymal invasion, appropriate steps must be taken to obtain specimens of highest quality for microbiologic testing.

Expectorated sputum is the specimen most frequently obtained for the laboratory diagnosis of lower respiratory tract infection. The importance of proper sputum collection was documented by Laird 100 years ago in studies on the yield of Mycobacterium tuberculosis according to the appearance and cellular composition of the sputum examined. The first requirement for collection of a good-quality sputum specimen is an alert and cooperative patient who can be instructed to rinse out his or her mouth with water or even brush his or her teeth before producing a lower respiratory tract specimen. The patient then must be encouraged to cough deeply to expectorate a specimen of lower respiratory tract secretions. With some infections such as tuberculosis (TB), a larger sample volume can improve the sensitivity of culture. Specimens are to be collected in sterile, leakproof, screw-capped containers. Containers should be transported in a watertight plastic biohazard bag.

Although a single sputum specimen may be sufficient for establishing the diagnosis of an acute bacterial process, collection of a series of two or three sputum specimens obtained on one or two days is recommended for patients suspected of having mycobacterial infections. In patients with nonproductive cough or suspected mycobacterial, fungal, or Pneumocystis jirovecii infections, it may be helpful to induce sputum production with an inhaled aerosol of hypertonic salt solution (3% to 10%).

Once collected, the specimens should be rapidly delivered to the laboratory for processing to avoid overgrowth by contaminating flora, which can compromise microscopic detection and isolation of pathogenic bacteria. Penn and Silberman found that organisms observed microscopically on Gram-stained smears of sputum specimens and their relative numbers in cultures changed dramatically between processing within an hour of collection and processing after overnight refrigeration. Although there were no significant differences in the culture results between the immediate and delayed cultures in this study, the loss of reliable microscopic features had significant impact on the interpretation of culture results. Processing delay is particularly important for culture recovery of slow-growing mycobacteria. Specimens that are not sent to the laboratory for processing within 2 hours should be refrigerated for no more than 5 days. If refrigeration is not possible, samples should be treated first with equal volume of 0.6% cetylpyridinium bromide or 1% cetylpyridinium chloride in 2% sodium chloride, which reduces the survival of contaminating microorganisms while preserving the viability of M. tuberculosis for up to 8 days. Although the recovery of fungi is optimal from cultures of fresh specimens, most clinically significant fungi appear to survive storage of 16 days or longer. Specimens for viral cultures should be shipped refrigerated but not frozen, whereas specimens for chlamydial culture should be placed into sucrose phosphate medium and shipped frozen.

Although there is no universal agreement on the value of anaerobic culture, protected catheter brushes may be used to obtain samples for culture and identification of organisms causing anaerobic pleuropulmonary disease. It is essential to transport samples in an anaerobic vial to preserve the viability of anaerobic organisms.

For detection of respiratory viruses, nasopharyngeal specimens are preferred, although lower respiratory tract specimens may be necessary to detect viral infection of the lower respiratory tract. There are a number of methods for the collection of nasopharyngeal specimens, which includes flocked and traditional swabs, as well as aspirates and washes. Flocked swabs contain perpendicular arrangements of fibers with an open structure to create a highly absorbent thin layer capable of efficient uptake of respiratory samples and elution into viral transport media. Nasopharyngeal flocked swabs have been shown to be more sensitive for the detection of respiratory viruses than traditional swabs. In turn, nasopharyngeal aspirates or washes have been shown to be more sensitive than nasopharyngeal flocked swabs. However, the modest gains in sensitivity for detection of most respiratory viruses using aspirates or washes may be offset by the ease of nasopharyngeal specimen collection using flocked swabs. Oropharyngeal specimens are less sensitive than nasopharyngeal specimens, though the combination may increase respiratory virus detection. Oropharyngeal swabs may also be used for detecting Chlamydophila pneumoniae, M. pneumoniae, and Legionella species.

In patients who are critically ill, immunocompromised, or who cannot produce expectorate, one or more invasive approaches may be necessary to obtain diagnostic samples. Specimens may include endotracheal aspirates, pleural fluids, bronchoalveolar lavage (BAL), percutaneous lung aspirate, or lung biopsies. The use of BAL has also been expanded to include diagnosis of bacterial pneumonia, especially for nosocomial cases. In patients with CAP requiring admission to the hospital, use of protected catheter brush and BAL has been shown to provide microbiologic diagnoses that are not obtainable by noninvasive means, although there is little support for using these procedures to diagnose CAP. Although the results of cultures from protected catheter brushes and BAL specimens are quantitatively similar, Meduri and Baselski concluded that BAL specimens provided a larger and more representative sample of lower respiratory tract secretions than the protected catheter brushes, allowing microscopic analysis of the cytocentrifuged BAL fluid to identify the type of bacteria present and to demonstrate the presence of neutrophils with intracellular organisms. These procedures may also yield additional pathogens not obtainable by noninvasive approaches. Much work has also been done with the use of BAL for the diagnosis of ventilator-associated pneumonia, (see Chapter 34 ).

In children under 7 years of age with suspected TB, gastric aspirate is used as a surrogate for respiratory samples. Historically it has been recommended that the pH of gastric aspirate be neutralized with sodium bicarbonate before transport to the laboratory; however, a recent study suggests that neutralization of gastric aspirate may reduce the recovery of M. tuberculosis . Nasopharyngeal aspirates have also been used for diagnosis of TB, although the sensitivity of culture-confirmed TB is lower compared to induced sputum. Stool samples in children with pulmonary TB may become the specimen of choice if processing methods can be optimized to concentrate the tubercle bacilli.

Other specimen types that may aid in diagnosis of lower respiratory tract infection include whole blood for blood culture, serum for antibody and antigen testing, and urine for antigen testing. Blood culture is recommended in cases of severe pneumonia but is positive only up to 37% in CAP and in less than 25% in nosocomial pneumonia. It is important to note that a large blood volume (60 mL or three sets of blood culture bottles in adults) is necessary to maximize sensitivity of blood culture. Although routine blood culture systems have been shown to be highly sensitive for detection of candidemia and cryptococcemia, automated blood culture systems are insensitive for cultivation of monomorphic and dimorphic molds. Isolation of molds (and fastidious bacteria) from blood requires the lysis-centrifugation method (Isolator) or the use of enriched fungal medium bottles.

S. pneumoniae can be recovered from urine cultures in as many as 38% of patients with pneumococcal pneumonia. Urine may be tested for the presence of pneumococcal and Legionella pneumophila serogroup 1 antigens. Fungal antigen tests of urine are also available for diagnosis of histoplasmosis and blastomycosis. Antigen assays are discussed later in this chapter.

Specimen Adequacy

Clinical laboratories are mandated by accrediting agencies to monitor specimen quality and quantity, and to enforce rejection criteria when sample requirements are not met. Common causes for rejection include insufficient sample quantity, poor sample quality, and mislabeling of samples. For bacterial cultures, microscopic examination of sputum and endotracheal aspirate with Gram stain is used to screen samples for adequate quality. The presence of excessive squamous epithelial cells (>10 to 25 per low-power field) is indicative of oropharyngeal contamination and therefore grounds for rejection for bacterial culture ( Fig. 17-1 ). Although earlier criteria for the adequacy of sputum specimens for bacterial cultures also required the presence of polymorphonuclear leukocytes (neutrophils), the number of neutrophils in a sample is no longer used to evaluate specimen adequacy. Endotracheal aspirates are rejected if the screening Gram-stained smears show no organisms. For mycobacterial, fungal, and viral cultures, cytologic screening to determine specimen acceptability is not enforced, because contamination with commensals does not interfere with interpretation of the culture results. However, the presence of respiratory columnar epithelial cells has been shown to improve respiratory virus detection by direct fluorescent antibody (DFA) testing.

Gram stain of sputum specimens.

A, This specimen contains numerous polymorphonuclear leukocytes and no visible squamous epithelial cells, indicating that the specimen is acceptable for routine bacteriologic culture. B, This specimen contains numerous squamous epithelial cells and rare polymorphonuclear leukocytes, indicating an inadequate specimen for routine sputum culture.

(From Tille P: Bailey & Scott’s diagnostic microbiology , ed 13, Philadelphia, 2014, Mosby, Fig. 69-4.)

Microscopy

Microscopic examination of lower respiratory tract specimens offers a rapid approach to detection and identification of many pathogens. However, as discussed earlier, a major limitation of microscopic examination is that it cannot distinguish between infection, colonization, and contamination when the specimen is collected through the oropharynx. In addition, microscopy lacks sensitivity in specimens with less than 10 ⁴ CFU per milliliter. Microscopy does routinely provide valuable information on the quality of specimen and the type of inflammatory response present. Specimens demonstrating a preponderance of polymorphonuclear leukocytes, ciliated columnar epithelial cells, or alveolar macrophages with few, if any, squamous epithelial cells (<10 per low-power field) represent lower respiratory tract secretions. The presence of alveolar macrophages is a more specific marker of lower respiratory tract secretions than neutrophils and is more likely to be associated with a significantly lower incidence of oropharyngeal contamination. The finding of neutrophils with intracellular organisms is considered indicative of an active infectious process.

The Gram-stained smear is an essential and necessary part of evaluation of sputum and tracheal aspirates for determining the quality and acceptability of specimens for bacterial culture and for providing a rapid assessment of the most likely etiologic agent of the pneumonia. Although Gram stains might also suggest the presence of mycobacteria, fungi, and parasites, special stains should be ordered when those pathogens are suspected. The Gram stain also stains squamous epithelial cells, ciliated columnar epithelial cells, neutrophils, and alveolar macrophages, which are used for assessment of specimen quality and inflammatory response. Table 17-7 shows criteria used by the laboratory to interpret findings on Gram stain and report them to the physician. Although it is impossible to correlate every staining pattern to a particular pathogen, several Gram stain patterns are pathognomonic for a particular pathogen or clinical entity ( Table 17-8 ).

Table 17-7

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