Direct Visual Examination

Lower respiratory tract specimens can be examined by direct wet preparation for parasites and special procedures for Pneumocystis. Fungal elements can be visualized under phase microscopy with 10% potassium hydroxide, under ultraviolet light with calcofluor white, or using periodic acid-Schiff–stained smears.

For most other evaluations, the specimen must be fixed and stained. Bacteria and yeasts can be recognized on Gram stain. One of the most important uses of the Gram stain, however, is to evaluate the quality of expectorated sputum received for routine bacteriologic culture. A portion of the specimen consisting of purulent mate rial is chosen for the stain. The smear can be evaluated adequately even before it is stained, thus negating the need for Gram stain of specimens later judged unacceptable. An acceptable specimen yields fewer than 10 squamous epithelial cells per low-power field (100×). The number of white blood cells may not be relevant, because many patients are severely neutropenic and specimens from these patients will not show white blood cells on Gram stain examination. On the other hand, the presence of 25 or more polymorphonuclear leukocytes per 100× field, together with few squamous epithelial cells, implies an excellent specimen (Figure 1). Samples that contain predominantly upper respiratory tract mate rial should be rejected. Previously, only expectorated sputa were suitable for rejection based on microscopic screening. However, endotracheal aspirates (ETAs) from mechanically ventilated adult patients can be screened by Gram stain. Criteria used to reject ETAs from adult patients include greater than 10 squamous epithelial cells per low-power field or no organisms seen under oil immersion (1000×). In Legionella pneumonia, sputum may be scant and watery, with few or no host cells. Such specimens may be positive by direct fluorescent antibody stain and culture, and they should not be subjected to screening procedures. Conversely, sputum from patients with CF should be screened. A throat swab is an accept able specimen from patients with CF in selected clinical settings and should be processed in a similar manner as CF sputum. Staining of respiratory samples is useful and should be compared to culture results to reveal errors in procedures, specimen collection, and transport or specimen identification.

Respiratory secretions may need to be concentrated before staining. The cytocentrifuge instrument has been used successfully for this purpose, concentrating the cellular material in an easily examined monolayer on a glass slide. As an alternative, specimens are centrifuged, and the sediment is used for visual examinations and cultures. For screening purposes, the presence of ciliated columnar bronchial epithelial cells, goblet cells, or pulmonary macrophages in specimens obtained by bronchoscopy or BAL indicates a specimen from the lower respiratory tract.

In addition to the Gram stain, respiratory specimens may be stained for acid-fast bacilli with either the classic Ziehl-Neelsen or the Kinyoun carbolfuchsin stain. Aura mine or auramine-rhodamine is also used to detect acid fast organisms. Because they are fluorescent, these stains fluorescent superior already here comment only are more sensitive than the carbolfuchsin formulas and are preferable for rapid screening. Slides may be restained with the classic stains directly over the fluorochrome stains as long as all of the immersion oil has been removed carefully with xylene. All of the acid-fast stains will reveal Cryptosporidium spp. if they are present in the respiratory tract, as may occur in immunosuppressed patients. These patients are often at risk of infection with P. jiroveci. Although the modified Gomori methenamine silver stain has been used traditionally to recognize Nocardia, Actinomyces, fungi, and parasites, it takes approximately 1 hour of the technologist’s time to perform, is technically demanding, and is not suitable as an emergency procedure. A fairly rapid stain, toluidine blue O, has been used in many laboratories with some success. Toluidine blue O stains Pneumocystis, Nocardia asteroides, and some fungi. A monoclonal antibody stain is the optimum stain for Pneumocystis for less invasive specimens such as BAL and induced sputa.

Direct fluorescent antibody (DFA) staining has been used to detect Legionella spp. in lower respiratory tract specimens. Sputum, pleural fluid, aspirated material, and tissues are all suitable specimens. Because there are so many different serotypes of legionellae, polyclonal antibody reagents and a monoclonal antibody directed against all serotypes of Legionella pneumophila are used. Because of low sensitivity (50% to 75%), DFA results should not be relied on in lieu of culture. Rather, Legionella culture, DFA or urinary antigen, and serology should be performed for optimum sensitivity. See Chapter 35 for details regarding detection of Legionella spp.

Commercially available DFA reagents are also used to detect antigens of numerous viruses, including herpes simplex, cytomegalovirus, adenovirus, influenza viruses, and RSV. Commercial suppliers of reagents provide procedure information for each of these tests. Monoclonal and polyclonal fluorescent stains for Chlamydia trachomatis are available and may be useful for staining respiratory secretions of infants with pneumonia. A number of molecular amplification techniques for the direct detection of respiratory pathogens have been described; however, the sensitivity and specificity of these assays vary greatly from one study to another. Amplification assays are also available for the direct detection of Mycobacterium tuberculosis on smear positive specimens.

Rapid direct detection from respiratory samples is now available using nucleic acid-based methods. The xTAG Respiratory Viral Panel (RVP) (Luminex Corporation, Austin, TX), can be used for the simultaneous detection of influenza (four types), RSV, human metapneumovirus, and adenovirus from nasopharyngeal swabs. In addition, the FilmArray Respiratory Panel (BIOFIRE Diagnostics, Salt Lake City, UT ) is capable of detecting upper respiratory tract infections associated with coronavirus (four types) , adenovirus, influenza (five types), rhinovirus, parainfluenza virus (four types), enterovirus, human metapneumovirus, RSV, Bordetella pertussis, Mycoplasma pneumoniae, and Chlamydophila pneumoniae in approximately 1 hour directly from patient samples. Smaller molecular panels are also available such as the real-time multiplex amplification kit for influenza A, B, and RSV (Hologic-Gen-Probe, San Diego, CA). All of the previously mentioned methods are FDA-approved. In addition to these, there are a variety of research-use only and other molecular respiratory panels in clinical validation studies. It is important when considering the use of a molecular assay that the laboratory consider their patient population including severity of illness, immune status, and transplant histories.

Routine Culture

Most of the commonly sought etiologic agents of lower respiratory tract infection are isolated on routine media: 5% sheep blood agar, MacConkey agar for the isolation and differentiation of gram-negative bacilli, and chocolate agar for Haemophilus and Neisseria spp. Because of contaminating oral flora, sputum specimens, specimens obtained by bronchial washing and lavage, tracheal aspirates, and tracheostomy or endotracheal tube aspirates are not inoculated to enrichment broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including transtracheal aspiration) and protected bronchial brush are suitable for anaerobic culture; the latter must be done quantitatively for proper interpretation (refer to prior discussion). Transtracheal and percutaneous lung aspiration material may be inoculated to enriched thioglycollate as well as to solid media. For suspected cases of Legionnaires’ disease, buffered charcoal-yeast extract (BCYE) agar and selective BCYE should be inoculated. Plates should be streaked in four quadrants to provide a basis for objective semiquantitation to define the amount of growth. After 24 to 48 hours of incubation, the numbers and types of colonies are recorded. For Legionella cultures, colonies form on the selective agar after 3 to 5 days at 35° C.

Sputum specimens from patients known to have CF should be inoculated to selective agar, such as specific chromagenic agar, for recovery of S. aureus and selective horse blood–bacitracin, incubated anaerobically and aerobically, for recovery of H. influenzae that may be obscured by the mucoid Pseudomonas on routine media. The use of a selective medium for B. cepacia, such as PC or OFPBL agars, is also necessary.

For interpretation of culture results on those specimens contaminated by normal oropharyngeal flora (e.g., expectorated and induced sputum, bronchial washings), growth of the predominant aerobic and facultative anaerobic bacteria is reported. To ensure optimum culture reporting, conditions must be well defined in terms of an objective grading system for streaked plates. Finally, the clinical significance of culture findings depends not only on standardized and appropriate laboratory methods but also on how specimens are collected and transported, other laboratory data, and the patient’s clinical presentation.

Numerous bacterial agents that cause lower respiratory tract infections are not detected by routine bacteriologic culture. Mycobacteria, Chlamydia, Nocardia, Bordetella pertussis, Legionella, and Mycoplasma pneumoniae require special procedures for detection; this also applies to viruses and fungi. Optimal recovery for Mycobacterium tuberculosis requires multiple specimens for acid-fast staining culture, and at least one sample for molecular testing as recommended by the Centers for Disease Control. Refer to the appropriate chapter section for more information regarding these organisms. Finally, one must keep in mind those potential agents for bioterrorist attack, such as Bacillus anthracis, Francisella tularensis, and Yersinia pestis, that might be recovered from respiratory specimens.

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مواضيع ذات صلة

tuberculosis testing (TB testing, Interferon gamma release assay [IGRA, T-Spot.TB], QuantiFERON-TB Gold [QFT, QFT-G, TB Gold test, TB blood test], Nucleic acid amplification for TB [NAAT], TB antibody)

thyrotropin-releasing hormone stimulation test (TRH stimulation test, Thyrotropin-releasing factor stimulation test [TRF stimulation test])

Laboratory Diagnosis of Lower Respiratory Tract Infections: Specimen Collection and Transport

thyroid scanning (Thyroid scintiscan)

thyroid fine needle aspiration biopsy (FNAB, Skinny needle thyroid biopsy, Fine-needle aspiration [FNA])

thoracentesis and pleural fluid analysis (Pleural tap)

The Role of Laboratory in Urgency: Altered State of Consciousness

Preoperative Laboratory Test

sputum culture and sensitivity (C&S, Culture and Gram stain)

sodium (Na), blood

small intestinal bacterial overgrowth test (SIBO test, Lactulose Breath Test)

Clinical Biochemistry of Exercise: The Benefits of Movement