General Thoracic Surgery (General Thoracic Surgery (Shields)) [2 VOLUME SET]

Editors: Shields, Thomas W.; LoCicero, Joseph; Ponn, Ronald B.; Rusch, Valerie W.

Title: General Thoracic Surgery, 6th Edition

Copyright 2005 Lippincott Williams & Wilkins

> Table of Contents > Volume I - The Lung, Pleura, Diaphragm, and Chest Wall > Section IV - Diagnostic Procedures > Chapter 14 - Laboratory Investigations in the Diagnosis of Pulmonary Diseases

Chapter 14

Laboratory Investigations in the Diagnosis of Pulmonary Diseases

Phillip G. Robinson

The purpose of this chapter is to introduce the thoracic surgeon to the diagnostic techniques that are available from the pathology laboratory. The chapter is divided into an anatomic and a clinical section. The anatomic section deals with surgical pathology and cytology specimens (Table 14-1). The section also discusses the different types of specimens, how they should be handled, and different examining techniques. The clinical section deals with infectious agents (Table 14-2) and the laboratory techniques available to detect them. Rosai(1996a,1996b) and Colby and associates (1994) summarized the special techniques in surgical pathology as well as the methods for handling specimens.

ANATOMIC SPECIMENS

The different types of anatomic specimens are listed in Table 14-1. They range from diagnostic biopsies to specimens resected for therapeutic purposes. The types of specimens vary from pleural biopsies to pneumonectomies, and they vary in size from several millimeters to an entire lung. To determine the proper specimen, the surgeon, radiologist, and pulmonologist should discuss the case and then consult with the pathologist before surgery.

Approach to Lung Biopsies

Lung biopsies are done for a variety of reasons that include tumors, interstitial lung diseases, and pulmonary infiltrates. The methods of obtaining specimens and the types of specimens are varied (see Table 14-1). Depending on the patient's clinical course, the suspected diagnosis, and the location of the lesion, the transbronchial biopsy is the procedure of choice, because of its low morbidity and cost. If the lesion is a solitary nodule and is out of reach of a bronchoscope, the transthoracic fine-needle aspiration or needle biopsy would be the next procedure of choice. The complications from bronchoscopy and needle biopsy or aspiration most frequently include pneumothorax or hemorrhage.

Thurer and I (1992) described the indications for an open lung biopsy. Candidates for this procedure usually have a rapidly progressive disease that takes the form of diffuse, persistent pulmonary infiltrates. The suspected diagnoses in these patients include infection, sarcoidosis, and lymphangitic carcinomatosis. In patients with acquired immunodeficiency syndrome (AIDS), open lung biopsies are used less and less frequently. Those patients can generally be managed by bronchial lavage, transbronchial biopsy, and in some cases, sputum examination.

Steinberg and associates (1998) found open lung biopsies to be an important tool in pediatric patients. Twenty-six children underwent biopsy, and the biopsies were diagnostic in 25 patients (96%). Therapeutic changes were instituted in 18 patients (69%) based on the results of the biopsy. In another study, Dai and associates (2001) evaluated open lung biopsy in seven patients with hematologic malignancies and progressive diffuse pulmonary infiltrates. Two patients had infectious processes, whereas the other five had diseases, such as alveolar proteinosis, idiopathic interstitial pneumonitis, leukemic involvement, and drug-induced alveolar damage. Two of three patients with acute lymphoid leukemia survived because of changes in their therapy secondary to the open lung biopsy. In summary, with all other biopsy modalities failing, the open lung biopsy can prove to be a valuable tool in evaluating and treating the patient.

Pleural Biopsy

The thoracic cavity contains two pleura. The visceral pleura covers the lung, and the parietal pleura lines the thoracic cavity. Pleural biopsies come from the parietal pleura.

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Transbronchial Biopsy

Transbronchial or endobronchial biopsies are usually obtained through a fiberoptic bronchoscope, although they may be obtained through a rigid bronchoscope. Since the introduction of the fiberoptic bronchoscope, the use of the rigid bronchoscope has declined. According to Mark (1984), fiberoptic bronchoscopes can visualize up to six generations of bronchi, whereas a rigid bronchoscope is useful for central lesions that involve the trachea and the two main bronchi. The transbronchial biopsy specimens are composed of four to five pieces of tissue that are submitted in formalin, with each piece measuring 1 to 2 mm in greatest dimension. Serial sections are performed on the block because the lesion may not be present on the first slide. Unstained sections may be made when the block is first cut because, owing to the small size of the specimen, it is technically difficult to go back and make additional sections. The unstained sections

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Chechani (1996) described a series of 49 patients with no airway abnormalities who underwent 51 fiberoptic bronchoscopic procedures to diagnose lung lesions. By using various sampling techniques that included brushing, transbronchial lung biopsy, bronchial washings, and Sofcor transbronchial needle aspiration, he established a diagnosis in 36 (73%) of the patients. He compared the diagnostic yield with the lesions' radiographic characteristics, size, and location in the lung as well as the number of sampling techniques used. He found that lesions with sharp borders had a lower diagnostic yield than those with irregular borders. A larger lesion yields a diagnosis more frequently than a smaller one. If the lesion was located in the basal segments of the lower lobe or the apical segments of the upper lobe, the chance of establishing a diagnosis was lower. Finally, if all of the aforementioned sampling techniques could be used, the chance of establishing a diagnosis was higher.

Churg (2001) editorialized on transbronchial biopsies and found that they produced reasonably high yields of diagnoses when the lesion involved was a malignancy, infection, sarcoidosis, or acute transplantation rejection. All of these situations provide specific diagnostic features. The biopsy is less likely to yield a specific diagnosis when the lesion is widely scattered throughout the pulmonary parenchyma, even if it has specific histologic features. Examples of these lesions are eosinophilic granuloma, lymphangioleiomyomatosis, and obliterative bronchiolitis in transplant recipients. Transbronchial biopsies are not useful for diseases that require examination of the low-power architecture of the lung, such as interstitial pneumonias and related conditions. Rather, an open lung biopsy should be performed.

Lung Needle Biopsy

Lung needle biopsies and aspirations are obtained under radiographic [computed tomographic (CT)] guidance and are directed at a specific lesion. The techniques for obtaining lung biopsies and aspirations are very similar, and the basic technique is described subsequently. Klein and Zarka (2000) reviewed the use of the transthoracic needle biopsy, including the indications, contraindications, imaging modalities, and biopsy techniques. The most common indication is to evaluate a solitary pulmonary nodule; other indications are to confirm metastatic disease to the lung, diagnose a mediastinal mass, diagnose pleural thickening, sample possible infectious nodules for cultures, and stage lung cancer. The contraindications are a bleeding diathesis, uncooperative patient, contralateral pneumonectomy, severe emphysema, and a vascular structure in the pathway of the needle.

Most biopsies are performed under CT guidance. The CT images allow the radiologist to place the needle in the tumor module. The coaxial needle placement technique is commonly used. A guide needle of about 19 gauge is inserted into the lesion. The stylet is removed, and either a cutting needle or an aspiration needle is placed into the lesion. A common cutting needle is a spring-loaded one with either an end-cut or side-notched device [e.g., Biopty (Bard Reynosa, S.S. de C.V., Reynosa, Mexico), ASAP-18 (Medi-tech, Boston Scientific Group, Natick, MA), Temno bone needle (Bauer Medical, Clearwater, FL)]. Once the biopsy needle is in the lesion, it is fired. Gazelle and Haaga (1991) reviewed the characteristics of the various needles.

The needle biopsies usually produce a specimen that consists of between one and three cylindrical fragments of tissue that measure about 10 mm in length by 1 mm in diameter. Like transbronchial biopsies, needle biopsies are so small that additional sections are usually cut when the slides are first made. The first slide should be stained with hematoxylin and eosin. After reviewing that slide and gaining an impression of the pathologic process, the pathologist can determine what stains to perform on the remaining slides. Table 14-3 lists stains that are useful for infectious agents, and Table 14-4 lists immunohistochemical stains that are useful for neoplasms. Specific immunohistochemical stains are described later in this section. The immunohistochemical technique is described in greater detail in Chapter 160. Needle biopsies of the lung are useful in determining the nature of a pulmonary lesion, and the results can guide the most appropriate therapy.

Connor and colleagues (2000) reviewed 106 transthoracic needle biopsies performed on 103 patients for their diagnostic accuracy and complication rates. If a diagnosis could not be established on the transthoracic needle biopsy, the patient underwent another procedure until the diagnosis was established. Eighty-five patients had malignant lesions, of which 75 (88%) were diagnosed on biopsy. Eighteen patients had benign lesions, of which 12 (67%) were diagnosed on biopsy.

As for complications, Connor and colleagues (2000) reported a 19% overall pneumothorax rate, but only 2.4% of those pneumothoraces were large (>30%) requiring chest drainage. As for hemorrhage, only 3.8% of the patients reported hemoptysis. Charig and Philips (2000) also examined the complication rate of transthoracic needle biopsies. Of 183 patients who underwent 185 procedures, 48 (25.9%) developed pneumothoraces. Of these patients, one required aspiration, and four required a chest tube. As for hemoptysis, 13 (7%) of the patients without pneumothorax developed it. Of these patients, only one was admitted for treatment. Charig and Phillips (2000) concluded that the complication rates for needle biopsy and needle aspiration were similar and that needle biopsy could be performed on an outpatient basis.

Open Lung Biopsy

The open lung biopsy or the video-assisted thoracic surgery (VATS) biopsy provides much more pulmonary tissue for examination than either the transbronchial or the needle

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The appropriate biopsy site depends on the location of the lesion. Gaensler and Carrington (1980) recommended avoiding taking a biopsy from the lingua. They believe that it normally shows scarring and chronic inflammation, which makes it difficult to diagnose interstitial lung disease. Wetstein (1986) and Miller and associates (1987), however, see no reason to avoid a biopsy of the lingua.

Frozen Section

The specimen for anatomic pathology should be examined by the pathologist before the end of the operation. A frozen section ensures the surgeon of an adequate specimen and provides a preliminary diagnosis. The tissue should be approached with a fresh blade. A sharp blade will minimize the crush artifact. At the time of frozen section, the pathologist can determine what other steps can be taken to arrive at the proper diagnosis. For instance, tissue can be placed in glutaraldehyde for electron microscopy, and this tissue should measure less than 1 mm. In many instances, immunohistochemistry has replaced electron microscopy in tumor diagnosis. Electron microscopy, however, may be useful in detecting the electron-dense material of immune complexes or amyloid. Tissue may also be placed in Michelle's solution for immunofluorescent studies. These studies would be useful for such diseases as Goodpasture's syndrome. Tissue may be frozen for special studies, such as immunohistochemistry, gene rearrangement studies, or some other study, but most of these studies can now be performed on formalin-fixed paraffin-embedded tissue. Air-dried and fixed (95% alcohol) touch preparations can be prepared.

If the frozen section suggests a lymphoma, tissue can be placed in RPMI media (a tissue culture medium), and immunophenotyping studies can be performed. If the lesion is a lymphoma or a plasma cell tumor, the immunophenotyping should yield a monoclonal population of cells. In addition, tissue may be placed in hematopathology fixatives that yield good nuclear detail, such as zinc-formalin, B5, Zenker's solution, or Bouin's solution. The remaining tissue should be placed in routine (10%) formalin solution. According to Churg (1983), a small syringe with a small-gauge needle can be used to inject formalin into the specimen. If the specimen has been cut, it may help to reduce the crush artifact. If the specimen does not have to be examined for a frozen section, the injection of formalin will both preserve

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Segmentectomy, Lobectomy, and Pneumonectomy

After the open lung biopsy, the larger pulmonary specimens include segmentectomies, lobectomies, and pneumonectomies. These larger specimens are usually the result of a therapeutic maneuver rather than a diagnostic one. Consequently, they are handled differently from an open lung biopsy. If the lung resection was for a tumor, the pathologist may be called to perform a frozen section on the bronchial margin. The frozen section ensures the surgeon that his margin is free of tumor. If indicated, the pathologist may also place tissue in RPMI for drug resistance studies or in special fixatives (see Fig. 14-1).

Corrin (2000) describes how the lung may be inflated with formalin. This is accomplished by placing a rubber tube into the main bronchus and securing the tube in place. The tube is connected to a container of formalin. The formalin is allowed to flow into the lung or lobe at a height of about 25 cm. After the lung is inflated, the tube is removed, and the bronchus is clamped with a hemostat. The specimen is placed in a deep pan filled with formalin. If the lung floats, it is covered with gauze or paper towels, which touch the formalin and serve as a wick. The surface of the specimen remains moist.

After about 4 hours, the specimen should be sufficiently fixed to be cut. Cutting or opening the specimen may be done in a variety of ways. The most common is by opening the bronchi with a pair of scissors. Corrin (2000) describes a method popularized by the late A. A. Leibow from Yale. A long probe is inserted down the bronchus until it perforates the visceral pleura. With the probe serving as a guide, a knife slices off the lung tissue above the probe. The cut lung tissue is removed, and this exposes the bronchial tree. This method can also be used as a lobectomy specimen. The segmentectomy specimens are small, and it may be easier to dissect out the bronchi with a pair of scissors.

CYTOLOGY SPECIMENS

Pulmonary cytology specimens vary considerably in type (see Table 14-1). The specimens range from the exfoliated cells of sputum to the retrieved specimens of lavages, brushings, and fine-needle aspirations. Pleural fluid is a form of retrieved specimen. All cytologic specimens are processed for a cell block and a cytospin preparation. A cytospin preparation consists of placing a small amount of well-mixed fluid (pleural, sputum) in a cytocentrifuge and centrifuging the cells onto a glass slide. The cells are usually stained with Papanicolaou's, Wright-Giemsa, and hematoxylin and eosin stains. The cell block is processed in a similar manner to tissue, and a histologic section is prepared from the block. The cell block and the cytospin are examined for malignant cells or whatever else the clinician suspects. Special stains and immunohistochemistry can be performed on the cell block.

Pleural Fluid

The pleural cavity normally contains a small amount of fluid that serves as a lubricant between the chest wall and the lung. Smith and Kjeldsberg (2001) divide pleural fluids into transudates and exudates. Transudates are due to increased hydrostatic pressure and usually occur in the setting of congestive heart failure. Exudates are due to increased capillary permeability or decreased resorption. They may be due to infections, neoplasm, pulmonary infarcts, or other pleural diseases. Pleural fluid must be sent for appropriate studies, which can include cultures, chemistry, cell count, and cytologic examination. Cytologic examination includes a cytospin preparation and a cell block.

Sputum

Sputum is a mixture of mucus, exfoliated cells, and alveolar macrophages, but it may also contain inflammatory cells, squamous cell (oropharyngeal contamination), and debris. An adequate specimen must contain alveolar macrophages. Sputum is used to diagnose pneumonia or a malignancy. For pneumonia, the sputum is sent to microbiology for culture and Gram's stain. For a malignancy, the sputum is sent to the cytology laboratory where the specimen is cytocentrifuged and a cell block made.

Cytologic examination of the sputum may be helpful. Because sputum is a passively acquired specimen, it is used for diagnosing malignancies. However, with the advent of bronchoscopy and fine-needle biopsy and aspiration, it is used less and less to diagnose malignancies. Sputum cytology was found to be ineffective in a National Cancer Institute trial for early lung cancer detection. In contrast, Petty (2000) describes a community lung cancer detection program in which sputum cytology examinations were useful in detecting lung cancer in its early stages in high-risk patients.

Bronchoalveolar Lavage

Bronchoalveolar lavage (BAL) specimens are useful in identifying both tumors and infections. Baughman and colleagues (2000) described BAL as wedging the bronchoscope into a distal airway and then injecting aliquots of normal saline into the lung through the suction channel of the bronchoscope. The amount of fluid injected and recovered is recorded. The fluid is pooled and then divided

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The fluid from a BAL should be processed in a standardized way. A cell count should be performed with a hemocytometer. Cytology should be performed as described for the cytology specimens. In addition to the stains mentioned, the Diff-Quik (Baxter Diagnostics, Inc., McGaw Park, IL, U.S.A.) may also be used. The BAL fluid may be used for a variety of chemical analyses. The specimens should be processed as rapidly as possible, which is usually within 4 hours. Otherwise, the samples should be stored at 4 C, and they may be kept for up to 24 hours. With delayed processing, the cell counts may be decreased, but the differential is usually the same.

Reynolds (2000) reviewed the use of BAL. The purpose of BAL is to study the pathologic processes in the lung. In the 1970s, the technique was used to study interstitial lung diseases, such as chronic hypersensitivity pneumonitis and idiopathic pulmonary fibrosis, which is known as cryptogenic fibrosing alveolitis in Europe. The BAL fluid of chronic hypersensitivity pneumonitis shows alveolar macrophages with a foamy cytoplasm, a high percentage of lymphocytes (60%), mostly T cells, a high immunoglobulin G (IgG)-to-albumin ratio, and IgM, which is not detected in patients with idiopathic pulmonary fibrosis. In contrast, the BAL fluid in patients with idiopathic pulmonary fibrosis contains neutrophils and eosinophils. The early uses of BAL fluid were for monitoring disease activity and researching interstitial lung diseases. Subsequently, BAL fluid never lived up to its expectations as a clinical laboratory test for interstitial lung disease.

Reynolds (2000) pointed out that by the late 1980s, BAL fluid was being used to recover microbial organisms, especially Pneumocystis carinii in AIDS patients, as well as to study other diseases, such as asthma, occupational lung diseases, acute lung injury, and the complications of organ transplantation. Future uses of BAL fluid analysis for the diagnosis of interstitial lung disease may be in conjunction with high-resolution computed tomography (HRCT). In summary, the BAL fluid provides a specimen from an area of the lung that is either involved with or very close to a diseased area, and the fluid is helpful in analyzing the disease process. BAL fluid analysis serves as a complement to current diagnostic studies, but does not replace them.

As reported by Goldstein and associates (1990), the American Thoracic Society pointed out that BAL is useful for making a diagnosis of carcinoma or metastatic carcinoma as well as other diseases, such as alveolar proteinosis, eosinophilic granuloma, eosinophilic pneumonia, and certain pneumoconiosis. Linder and colleagues (1987) described using BAL to diagnose cancer. They described 35 cases of biopsy-proved lung carcinoma, in which the BAL showed malignant cells in 24 (68.6%) of the patients. The agreement between the cancer subtypes from the BAL and the biopsy was 79.1%. The variations occurred between large cell undifferentiated carcinoma and adenocarcinoma, but not small cell carcinoma. Although lung carcinomas can be diagnosed by BAL, not many reports exist in the literature. As the aforementioned study shows, a biopsy gives a more specific histologic diagnosis, and the BAL may not be positive in all cases.

In contrast, BAL is very useful in managing lung transplant recipients. Tiroke and colleagues (1999) described the normal BAL findings in a transplanted lung as well as the abnormal findings in a reimplantation response, acute and chronic rejections, and infections. The reimplantation response occurs in the first week after transplantation, and the BAL contains a markedly increased number of neutrophils. The reimplantation response is a reversible organ dysfunction, probably brought about as a response to ischemia and reperfusion. Acute rejection usually occurs in the first week after transplantation and is characterized by an increased cell count in the BAL and lymphocytosis. Chronic rejection is characterized by obliterative bronchiolitis. This diagnosis can only be made on tissue biopsy. The BAL, in chronic rejection, shows an increased cell count with both lymphocytes and neutrophils. In the proper clinical setting, this cytology is suspicious for a chronic rejection.

Bronchial Brushing

Metersky and Gupta (1998) described bronchial brushing as passing a sheathed brush down a flexible bronchoscope and then passing the brush over the lesion. Cells or organisms are trapped in the bristles of the brush. The sheathed brush is then pulled through the bronchoscope. The cells on the brush are smeared out on glass slides. Some slides are immediately placed in 95% alcohol, and others are allowed to air dry. Sato and colleagues (2002) found that brushes with bristles that measured less than 0.1 cm in diameter collected more cells and showed less air-drying artifact. Gaber and associates (2002) performed bronchial biopsy, conventional brushing, lavage, and whole endobronchial brushing on 39 patients with endoscopically visible lung tumors. The whole endobronchial brush was cut off and placed in Shandon's

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Lung Fine-Needle Aspiration

Fine-needle aspirations of the lung are performed in a manner similar to transthoracic needle biopsies. The technique for transthoracic needle biopsy was described earlier. Klein and Zarka (2000) described the technique for needle aspirations. The aspiration needle can be inserted into the lesion either through a guide needle or by itself. After the imaging studies confirm the presence of the needle in the lesion, a 10- or 20-mL syringe is attached to the needle or to a plastic tube that is attached to the needle and then aspirated. The needle is usually moved in an up-and-down or rotary motion, piercing the lesion multiple times, during which suction is applied. After 2 to 5 seconds, the needle is removed, and the aspirated tissue is placed in normal saline or smeared out on microscopic slides. If a guide needle is present, the aspirate can be performed more than once. The fluid from the tip of the needle is smeared on the slides. The slides are both air dried and immediately fixed in 95% alcohol. The needle, tubing, and syringe are rinsed with physiologic saline. The fixed smears are stained with hematoxylin and eosin and Papanicolaou's stains, whereas the air-dried smears are stained with Wright-Giemsa stain. The material in physiologic saline is cytocentrifuged to produce cytospin smears. If sufficient tissue is present, the remaining fluid can be used to make a cell block. Transbronchial needle aspirations can also be performed with the material processed in a similar manner. They have the same complications of pneumothorax and hemorrhage as needle biopsies of the lung.

The value of transthoracic fine-needle aspirations as opposed to transthoracic needle biopsies is uncertain. Greif and colleagues (1998) compared the result of transthoracic needle biopsies with fine-needle aspiration from 156 patients. Both biopsies were performed sequentially at the same visit. The aspiration provided a diagnosis in 133 (85.3%) of the cases, and the needle biopsy provided a diagnosis in 121 (77.6%) of the cases. The needle biopsy confirmed the aspirate in 90 (57.7%) patients, provided additional information in 17 (10.9%) patients, and was less informative than aspiration in 35 (22.4%) patients. They concluded that needle biopsies offered no advantage over aspiration for peripheral lung lesions. In contrast, Yilmaz and associates (2001) performed fine-needle aspirations in 129 patients with lung nodules who subsequently underwent a thoracotomy. They found a concordance of 73.6% between the aspiration diagnosis and the thoracotomy diagnosis. The poorest agreement was for large cell carcinomas. If the tumors were well differentiated, there was a high degree of agreement between the aspirate and the tissue. They concluded that aspirations did not always lead to the correct tumor types. In one final study, Delgado and co-workers (2000) found that aspirations were highly sensitive in distinguishing small cell carcinoma from non-small cell carcinoma. They concluded that aspiration is useful in selecting the appropriate modality to treat lung neoplasms.

SPECIAL STAINS

Special stains defy a neat classification. Anatomic pathology has a certain degree of subjectivity that requires a judgment on the part of the pathologist. The purpose of special stains is to confirm a morphology judgment and to make that judgment less subjective. Special stains can be divided into nonimmunologic stains and immunologic stains. The nonimmunologic stains can be for organisms (see Table 14-3) or for other purposes, such as a trichrome stain for collagen.

Immunologic staining techniques are more objective, sensitive, and specific. Immunohistochemical stains involve the binding of either a monoclonal or a polyclonal antibody to a specific antigen and then visualizing the reaction. An example would be the binding of the antibody known as CAM 5.2 to low-molecular-weight cytokeratins. If the tumor cells immunostain for keratin, the pathologist has objective evidence that the tumor is a carcinoma. Coons and associates (1941) first used this technique, and their antibody was labeled with fluorescin isothiocyanate. Fluorescin proved a poor marker for general use and was replaced by the peroxidase-antiperoxidase method and the biotin-avidin method described by Hsu and associates (1981). These techniques are discussed in Chapter 160 as well as by Rosai (1996a) and Taylor and Coates (1994). Table 14-4 lists useful immunohistochemical stains for pulmonary tumors.

Routine Special Stains

Routine special stains can be divided into those for microorganisms and those for other purposes, such as an iron stain. This section focuses on special stains for microorganisms, and these are listed on Table 14-3. The Ziehl-Neelsen stain for mycobacteria and the Grocott-Gomori methenamine silver (GMS) stain for fungus are probably the most commonly used. The Ziehl-Neelsen stain makes mycobacteria red, and Mycobacterium tuberculosis has the appearance of a beaded rod. GMS stains the walls of fungi and Pneumocystis species black. Cryptococcus species stain black with GMS, but they also have a mucinous capsule that will stain pink with a mucicarmine stain. Despite the advances in immunohistochemistry, these routine special stains are very useful. Taylor and Cotes (1994) reviewed

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Immunohistochemical Stains

The immunohistochemical stains in pulmonary diseases are most useful in identifying lung neoplasms (see Table 14-4). Tumors in the lung can be primary or metastatic. If the malignant cells are large, Gown and Bacchi (2000) use a panel of immunohistochemical stains that includes cytokeratin, vimentin, HMB 45 (a melanoma antigen), and CD45 (leukocyte common antigen). The cytokeratin identifies a carcinoma; the vimentin, a sarcoma; HMB45, a melanoma; and CD45, lymphomas. If the tumor proves to be a carcinoma, additional immunostaining can be performed to determine whether the tumor is a primary pulmonary tumor or a metastatic one.

Thyroid Transcription Factor-1

Zamecnik and Kodet (2002) describe thyroid transcription factor-1 (TTF-1) as a tissue-specific homeodomain-containing transcription factor of the Nkx2 gene family that is expressed in the lungs, thyroid gland, and some areas of the diencephalon during development. Following birth, the protein remains detectable in the thyroid and in Clara cells and type II pneumocytes. Zamecnik and Kodet (2002) immunostained 109 tumors for TTF-1. Fifty-four of the tumors were pulmonary carcinomas, and 55 were nonpulmonary carcinomas. Their results for the TTF-1 immunostaining are shown in Table 14-5 and are compared with the immunostaining results for an antibody against surfactant apoprotein A (SP-A, PE10), discussed subsequently. The table shows that TTF-1 immunostains 75% of the pulmonary adenocarcinomas, 50% of the pulmonary large cell carcinomas, and 89% of the pulmonary small cell carcinomas. In contrast, the pulmonary squamous cell carcinomas and the typical carcinoids do not immunostain with TTF-1. TTF-1 is a nuclear stain, and its staining is demonstrated in a primary pulmonary adenocarcinoma (Fig. 14-2). In the nonpulmonary carcinomas that Zamecnik and Kodet (2002) immunostained for TTF-1, only the thyroid carcinomas were positive, with the exception of the anaplastic thyroid carcinoma that did not immunostain for TTF-1.

Ordonez (2000) reviewed the usefulness of TTF-1 in discriminating between pulmonary small cell carcinomas and small cell carcinomas from other sites, such as Merkel cell carcinomas. He also used cytokeratin 20 (CK20, a cytokeratin) to help discriminate between small cell carcinomas from different sites. He found that small cell lung carcinomas were usually TTF-1 positive and CK20 negative. In contrast, Merkel cell carcinomas were TTF-1 negative and usually CK20 positive. This staining pattern could be helpful in identifying the source of a metastatic small cell carcinoma. Of interest is the TTF-1 staining of both the round and surface cells composing the sclerosing hemangiomas of the lung. Devouassoux-Shisheboran and collaborators (2000) proposed that this suggests the tumor's origin to be from primitive respiratory epithelium.

Cytokeratins 7 and 20

Cytokeratin 7 (CK7) and cytokeratin 20 (CK20) are keratin proteins that have a unique distribution among normal epithelial cells. Wang and associates (1995) found that non small cell carcinomas of the lung were CK7 positive and CK20 negative. Other tumors that show this staining pattern are breast carcinomas, ovarian carcinomas, mesotheliomas, endometrial adenocarcinomas, and some pancreatic carcinomas. Chhieng and colleagues (2001) found this staining pattern in 85% of lung carcinomas, but they also found it in 55% of nonpulmonary carcinoma specimens. They concluded that also doing an immunohistochemical stain for TTF-1 and surfactant apoprotein A (discussed subsequently)

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Surfactant Apoprotein A

Surfactant apoprotein A (SP-A) is a surfactant produced by alveolar type II cells and Clara cells. This apoprotein is identified by the antibody PE 10. Zamecnik and Kodet (2002) immunostained a series of lung tumors with an antibody against SP-A (PE-10). They found that 60% of the pulmonary adenocarcinomas and 25% of the pulmonary large cell carcinomas immunostained with SP-A (see Table 14-5). In contrast, none of the pulmonary squamous cell carcinomas, typical carcinoids, or small cell carcinomas immunostained with SP-A. They concluded that SP-A is not as useful as a marker for lung carcinomas as TTF-1.

MOLECULAR PATHOLOGY

The purposes of this section are to introduce the reader to some basic concepts of molecular pathology and how they apply to the laboratory and to discuss the terminology of molecular biology. Molecular pathology applies to both anatomic and clinical pathology. After the introduction, the remainder of the chapter focuses on specific pulmonary infectious agents and discusses some of the molecular methods used to diagnose them. The molecular biology of lung carcinoma is covered in Chapter 102. In anatomic pathology, most of the clinical testing for the molecular markers of carcinomas is done by immunostaining, which is described in Chapter 160.

TERMINOLOGY OF MOLECULAR BIOLOGY

Human cells contain 23 pairs of chromosomes. Of these pairs, 22 are autosomes, and one pair is the sex chromosomes, XX in the female and XY in the male. The chromosomes, located in the cell nucleus, are composed of DNA and encode the genetic information of the organism. Each chromosome bears a linear array of genes. The 22 autosomal chromosomes are paired, and each autosomal locus is represented twice. An allele is any particular form of a gene that can exist at a particular locus. If both chromosomes have the identical gene or allele at the same locus, the genes are referred to as homozygous alleles. If the genes are not identical at the same locus, they are referred to as heterozygous alleles.

DNA exists in the form of a double helix, and when it is transcribed into messenger RNA (mRNA), it unwinds. The transcription requires three RNA polymerases as well as proteins known as transcription factors. Santis and Evans (1999a, 1999b), in their review of molecular biology, point out that DNA and mRNA are not collinear. In other words, the mRNA is not complementary to the DNA because the DNA contains exons (expressed sequences) and introns (noncoding sequences). The introns are removed from the final sequence of the mRNA.

According to Santis and Evans (1999a), gene identification and mapping led to the recognition that individuals differ considerably in the structure of their DNA, particularly in the intron regions, and this difference is known as genetic polymorphism. This variation in DNA can be identified by molecular techniques, such as restriction fragment length polymorphism (RFLP), discussed later. According to Palmer and Cookson (2001), genetic polymorphism arises from mutations. The different types of polymorphisms are named on the basis of the type of mutation from which they come. The simplest class of polymorphism is a single base mutation that substitutes one nucleotide for another. This type of polymorphism is known as single nucleotide polymorphism and can be detected by RFLP.

MOLECULAR DIAGNOSTIC TECHNIQUES

Molecular pathology is defined as the study of biochemical and biophysical cellular mechanisms as the basic factors

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Electrophoretic Separation

The sugar-phosphate backbone of DNA and RNA has a negative charge that is proportional to the length of the chain. When an electric field is applied to the molecules, they separate according to their size. The molecules are usually separated in a medium known as a gel, and the composition of the gel determines the size of the fragments that can be separated. The most common medium is agarose, but others, such as sucrose and Ficoll, are also used. An example of electrophoresis in molecular biology is the Southern blot. Southern (1975) developed a technique to detect specific DNA sequences. His technique consisted of digesting the DNA with restriction endonucleases and then separating the DNA fragments by electrophoresis on agarose gel. The separated DNA fragments were denatured to single strands with a basic solution and transferred by blotting from the agarose gel to cellulose nitrate filter paper. The filter was heated, causing the single-stranded fragments of DNA to become permanently attached to it. The immobilized DNA was hybridized with a radiolabeled probe. After rinsing away the excess probe, the filter was autoradiographed on standard radiographic film. The developed film showed a series of bands corresponding to the location of the probed DNA. Figure 14-3 illustrates Southern's technique with reference to the rearrangement of immunoglobulin genes.

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Hybridization Assays

Hybridization is the interaction between two complementary single strands of nucleic acids whereby they unite to form a double strand, such as double-stranded DNA. When neither strand of DNA is labeled, the process is called annealing. If one strand is labeled, that strand is called a probe, and the process is called hybridization. The complementary pairing can occur between two strands of DNA, two strands of RNA, and one strand of RNA and one of DNA. The environmental conditions under which these pairings take place can be controlled as to temperature, salt, and formamide (a chemical used to denature nucleic acids). These conditions are known as the stringency under which the strands combine.

Hybridization assays are the basis for identifying bacteria, fungi, protozoa, or viruses either in clinical specimens (urine, stool, blood, sputum) or to confirm their presence from a culture. Hybridization assays are composed of four parts: the probe, a specimen, controlled conditions for pairing, and a system to detect the pairing. The probe is a single strand of either RNA or DNA that is complementary to a known nucleic acid sequence in a microorganism. The conditions under which the nucleic acids combine must be maintained at certain stringency. Finally, after the probe has hybridized, it must be detected. The first assays used radioactive labels, such as phosphorous 32 (³²P), that were detected through autoradiography or scintillation counting. Detection methods that use nonradioactive substances are very similar to those used for immunohistochemistry (see Chapter 160). The detection method may involve biotin, which then combines with avidin or streptavidin that is carrying a signal-generating enzyme or a fluorochrome.

Santis and Evans (1999b) pointed out that probe tests may be unamplified or amplified. Unamplified probes are less sensitive than the usual microbiology techniques and are used mostly for detection rather than diagnosis of organisms. Consequently, techniques exist to amplify either the target DNA or the hybridized probe. The target DNA is amplified by the use of the polymerase chain reaction (PCR). Abe and colleagues (1993) described the amplification of a repetitive sequence of M. tuberculosis by PCR in which they detected the organism in 84% (32 of 38 specimens) of culture-positive sputa.

Restriction Fragment Length Polymorphism

RFLPs are pieces of DNA with varying lengths. RFLPs are created because of restriction endonucleases and genetic polymorphism. A restriction endonuclease is a bacterial enzyme that repeatedly breaks DNA strands apart at the same specific nucleotide base sequence site. Hence, the enzymes yield reproducible fragments of DNA. Smith and Wilcox (1970) described the first restriction endonuclease enzyme, which they accidentally isolated from the bacteria Haemophilus influenzae. Since their initial work, many other restriction endonucleases have been described; they come from a variety of different bacteria. Restriction endonucleases are named for the bacteria from which they are isolated. For example, Bam H1 is derived form Bacillus amyloliquefaciens H and is active only at the base

sequence site, whereas others are active at other highly specific base sequence sites. After digesting the DNA strands, the fragments are separated on the basis of their size by agarose gel electrophoresis and visualized by staining with a DNA dye. The molecular weight of the fragments is determined by comparing their position on the gel to the position of DNA fragments with a known molecular weight.

Genetic polymorphism occurs because a gene for a trait may have a slightly different DNA sequence. According to Todd and colleagues (2001), these slightly different sequences may be due to natural variation (polymorphism) or genetic alterations (mutation, deletions). Housman (1995) pointed out that most of the polymorphism between genes occurs in the tandem-repeat sequences. These are segments that show a repetition of the same short DNA sequence a variable number of times. They may occur in the coding regions (exon) or in the noncoding regions (introns) of the DNA. Figure 14-3 illustrates the use of endonucleases with regard to evaluating the rearrangement of immunoglobulin genes by Southern blot. The first step in the process is to digest the DNA with an endonuclease.

Amplification

Amplification is an increase in the number of copies of a specific DNA fragment. The best known of the amplification techniques is the PCR. The following discussion focuses on this technique but also briefly explains the other techniques. Zimring and Nolte (2001) divided amplification methods into three types: target, probe, and signal. PCR belongs to the target type of amplification.

Target Amplification

Target amplification is an enzyme-mediated process that produces many copies of a nucleic acid. Three target amplification methods exist: PCR, transcription-mediated amplification/nucleic acid sequenced-based amplification (TMA/ NASBA), and strand-displacement amplification (SDA). This discussion focuses on PCR and its modifications. According to Persing (1991), PCR is a method to replicate small quantities of either RNA or DNA such that a sufficient quantity exists to be detected. PCR works in identifying microorganisms because each one possesses unique sequences of RNA and DNA.

PCR works by combining the following ingredients for the reaction, double-stranded DNA (dsDNA) of interest,

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Wolk and co-workers (2001) described the non-PCR target nucleic acid amplification methods, which are the second two listed previously. These techniques do not require a change in temperature (isothermal). They consist of TMA and NASBA, both of which are listed and both of which use an RNA target. Reverse transcriptase synthesizes a complementary DNA strand (cDNA). The cDNA becomes a template to synthesize multiple RNA copies with the enzyme RNA polymerase. The copies of RNA are detected.

SDA, as described by Wolk and colleagues (2001), consists of two primers, both of which contain a target-specific region and a hemimodified restriction enzyme site. A primer is a short segment of DNA that hybridizes with a single strand of DNA. Once the primer hybridizes, the DNA polymerase synthesizes the complementary strand. Both primers contain a restriction (endonuclease) enzyme site. When the restriction enzyme is added, it creates a nick at the restriction site. The DNA polymerase initiates a new strand, and the old strand is displaced without being destroyed. The process continues with nicking, extension, and displacement resulting in the amplification of the original DNA target.

Probe Amplification

Probe amplification amplifies only the DNA or RNA sequence of the original probe. A probe is a piece of DNA or RNA that corresponds to a gene or a nucleic acid sequence of interest. The probe is usually labeled radioactively or with a detectable molecule, such as biotin or fluorescein. A probe will hybridize to its complementary sequence. The two techniques for probe amplification are ligase chain reaction and Invader and Cleavase technology.

Tang and associates (1997) describe the process of ligase chain reaction. DNA is denatured into single strands. Two separate probes anneal to two separate areas of target DNA. These target nucleic acids are separated by only one to three nucleotides. A ligase is introduced into the solution, and the two probes are ligated together. Each probe has detection or a capture hapten attached to it. The ligated probes are separated from the target by heating. The process repeats itself with new probes hybridizing and being ligated. The denatured strands serve as templates. After the probes are amplified, they are detected through the attached capture hapten.

Wolk and colleagues (2001) describe the Invader technology (Third Wave Technologies, Inc., Madison, WI, U.S.A.) as isothermal signal probe amplification. The technique uses an Invader probe, a signal probe, and the enzyme Cleavase. The process starts with a denatured strand of DNA. The two probes are added, with the Invader probe binding upstream of the signal probe. The two probes overlap by at least one nucleotide. Cleavase recognizes the specific base pair overlap configuration of the two bound probes, and it cleaves the overlapping portion of the signal

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Signal Amplification

Signal amplification does not increase the number of either the target or the probe DNA or RNA. Signal amplification focuses on increasing the number of labeling molecules at the target sequence. The three techniques for signal amplification are branched DNA, hybrid capture, and Q-beta replicase.

Signal amplification increases the signal generated from the probe hybridized to the target nucleic acid. Wolk and associates (2001) give the following description of the branched DNA assay. The organisms, for example, hepatitis B virus (HBV), are disrupted in a well coated with capture probes to the DNA of HBV. The HBV DNA is then fixed to the well through the capture probes. Target probes are added to the solution, and they hybridize to viral DNA and to the signal amplifier. The signal amplifier is a piece of branched DNA with many binding sites for enzyme-labeled probes. It is added to the solution and binds to the target probe. Finally, enzyme-labeled probes are added to the solution and bind to the multiple sites on the branched DNA. The enzyme-labeled probes are detected by a chemiluminescent process.

The hybrid capture assay involves an RNA probe hybridizing to a DNA target, or the opposite, a DNA probe to an RNA target. The RNA:DNA hybrids are captured by antibodies bound to a well that are specific for the hybrid. An antibody with a chemiluminescent enzyme is then added to the well and binds to the hybrid. Finally, the enzyme is activated, with the released light being detected.

Lizardi and co-workers (1988) first described the Q-beta replicase system. The system is an isothermal signal amplification system that is based on the function of the enzyme replicase that is found in the RNA virus Q-beta. The technique involves an RNA probe, RNAse III, and the Q-beta replicase enzyme. The RNA probe is shaped like a cross, with the head containing the target-specific probe and the tips of the two arms containing the replicase substrate. The center of the cross contains the cleavage site for RNAse III. The probe hybridizes to a single strand of DNA. The RNAse III is added, and the excess probe is digested and then washed away. Q-beta replicase is added to the solution, and the hybridized probe is replicated to detectable quantities.

INTRODUCTION AND USES OF MOLECULAR TECHNIQUES IN MICROBIOLOGY

Bryant-Greenwood (2002) emphasized that traditional microbiology techniques are currently superior to molecular methods except in the case of fastidious organisms (Legionella pneumophila), slow-growing organisms (M. tuberculosis), and unculturable organisms (hepatitis C virus). Gilbert and colleagues (1999) add that molecular techniques also have the ability to offer automation, make specimen transportation less critical, and reduce the risk for laboratory-acquired infections. The current microbiology techniques of culture and susceptibility testing work well with fast-growing organisms, such as the gram-positive cocci and the gram-negative rods.

Tang and associates (1997) describe the conventional techniques for microbial identification as relying on microbial morphology, growth variables, and the phenotypic characteristics of the organisms. Current culture techniques allow for a variety of organisms to grow on the culture media or medium upon which they are inoculated. Molecular techniques focus on a single organism, although the future will most likely bring about a technique to probe for multiple pathogens that infect a particular body site. The technique will probably be trays with a series of wells, with each well containing a probe for a certain organism.

Hybridization studies were first used in microbiology to determine the relatedness of organisms. Subsequently, epidemiologists used molecular techniques to study outbreaks of infectious disease. The advent of amplification techniques, such as PCR (see Table 14-6), has made way for new techniques for detecting and characterizing microbes. With amplification techniques, the organism can be identified from a clinical specimen rather than being grown in a culture and then identified. For example, Paton and Paton (1998) used molecular techniques to detect toxin-encoding genes of enterohemorrhagic Escherichia coli. De Beenhouwer and associates (1995) used molecular techniques to detect multidrug-resistant M. tuberculosis by looking for known mutations of the rifampin-resistant genes. In summary, molecular techniques are finding applications in microbiology in identifying organisms, tracing the epidemiology of an epidemic, identifying drug resistance, and identifying the presence of toxin-producing genes. Most of the techniques described in the molecular diagnostic techniques are being applied in the microbiology laboratory.

SEROLOGIC TECHNIQUES FOR ESTABLISHING PRESENCE OF INFECTION

Serologic methods generally detect either the antigens produced by an organism or the host antibody response to the organism. The organism antigen or the host response can be detected in a variety of ways. For example, a latex agglutination assay exists that detects cryptococcal antigen in the cerebrospinal fluid (CSF) of patients with cryptococcal meningitis. Quantification of antigen assays is not imperative because the presence of the organism or its antigen establishes the presence of an infection. In contrast, most of the population is infected with the herpesvirus, and the

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The patient's antibody response is quantitative and serves to distinguish a past infection from a current one. Quantification is usually performed by serial dilutions that are reported as titers (i.e., 1:4, 1:256). Active infection usually results in high titer, such as 1:256, or an increase in titer, typically fourfold or greater in a patient previously exposed to the organism. Two time points are necessary to make this determination, with serum being drawn at the time of acute infection and later during the convalescence phase, about 6 to 8 weeks, for comparison. Furthermore, assay of the class of antibody can be important because the IgM class of antibodies appears early in the infection and disappear after about 6 months. In contrast, the IgG class antibodies appear later in the infection and persist for years, indicating a previous exposure to the microbial antigen.

A daunting number of assays are used for the evaluation of the humoral response to microbial infection. The most commonly performed include complement fixation, indirect hemagglutination, direct and indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and immunodiffusion. The relative merits of each of these specific techniques are not particularly important to the clinician except insofar as they all represent tools to evaluate the humoral status of the patient in regard to a specific microbe.

SPECIFIC LABORATORY IDENTIFICATION OF VARIOUS INFECTIOUS AGENTS

The remainder of the chapter discusses the current techniques for identifying specific microorganisms. The organisms include bacteria, L. pneumophila, Nocardia species, mycobacteria, fungi including Pneumocystis jiroveci, parasites, and viruses with emphasis on the family of herpesviruses (see Table 14-2).

Legionella pneumophila

In July 1976, a strange and virulent form of pneumonia struck 182 members of the Pennsylvania branch of the American Legion during their annual meeting in Philadelphia; 18 legionnaires died. During the next 6 months, an intensive investigation resulted in the isolation of a new, completely different bacterial organism, now known as L. pneumophila. This organism differs from other medically significant bacteria in that it does not stain by Gram's method and is unique in having an absolute growth requirement for cysteine. An adequate amount of this compound to grow Legionella is not present in blood agar, chocolate agar, or other types of primary culture media. The organism also requires an increased concentration of CO₂ for growth and may take 7 to 12 days to appear on artificial culture media. The organism grows best on buffered charcoal yeast extract, which contains L-cysteine and iron salts, supplemented with -ketoglutarate.

Stout and co-workers (1982) showed that the isolation of L. pneumophila and related species initially from the water in air-conditioning cooling towers and subsequently from the faucets, shower heads, and hot-water storage tanks in hospitals emphasizes the ubiquitousness of this group of organisms. Because the organisms are widespread, it is clear that mere exposure to contaminated water is an insufficient condition for the occurrence of legionnaires' disease; host susceptibility is undoubtedly a critical factor. Patients undergoing immunosuppression for organ transplantation or other therapeutic reasons are at high risk and should be followed carefully for the sudden onset of severe and rapidly developing pneumonia.

Clinically, the disease may occur in epidemics, such as was seen in Philadelphia, or as sporadic cases acquired in the community. As reported at the International Symposium on Legionnaires' Disease (1979), it has been recognized as a major cause of serious or fatal pneumonia in immunosuppressed or immunodefective patients, many of whom develop the disease while in the hospital.

The laboratory diagnosis of Legionella is based on rapid methods, culture, and serology. The serologic diagnosis of Legionella depends on development of convalescent titers and thus is not useful for diagnosis of acute disease. An exception is the demonstration of high titers (i.e., 1:256 or greater) during the acute disease that is indicative of active infection, particularly because symptomatic carriage does not occur, and reinfection with Legionella does not appear to be common. Culture is the most specific diagnostic method, but it has limited sensitivity and requires at least 2 days and possibly up to 2 weeks.

To guide therapy, rapid methods for the diagnosis of Legionella have evolved. The technique most widely used is direct fluorescence antibody staining of sputum specimens; direct fluorescence antibody staining is rapid but suffers from a lack of sensitivity, usually about 60% to 75%. Other rapid methods include nucleic acid hybridization probes and detection of antigen in urine, sputum, or serum. Fain and co-workers (1991) developed a hybridization technique. Hayden and associates (2001) compared the detection of Legionella in BAL fluid and open lung biopsy using real-time PCR (Light Cycler, Roche Molecular Biochemicals, Indianapolis, IN, U.S.A.) in situ hybridization (ISH), direct fluorescence antigen detection, and culture. The BAL fluid was tested by direct fluorescence antigen detection (DFA) and real-time PCR. The DFA test was positive in 33% of the cases. The real-time PCR was positive in 100% of the cases. The open lung biopsy specimens were tested with DFA, ISH, real-time PCR, and Warthin-Starry staining. The DFA gave a positive test 44% of the time. The Warthin-Starry stain gave a positive result 63% of the time.

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Assays for Legionella antigen in urine, sputum, and serum samples include ELISA, latex agglutination, and radioimmunoassay techniques. Helbig and colleagues (2001) compared three urine antigen detection kits (Binax NOW, Binax EIA, and Biotest EIA). These kits detect the antigen of L. pneumophila serogroup I, which probably accounts for at least half of the infections. The Binax NOW is an immunochromatographic assay, whereas the other kits are enzyme immunoassays. The Binax NOW and Binax EIA are only for the detection of L. pneumophila serogroup I, whereas the Biotest EIA is for the detection of all serogroups. Of the 187 Legionella infections confirmed by culture or seroconversion, or both, 163 (87.2%) were positive by at least one urinary antigen assay, but only 74.3% were positive in all of them. Consequently, Helbig and colleagues (2001) concluded that direct fluorescence antigen detection and culture were still important tools in diagnosing L. pneumophila infections. Waterer and co-workers (2001) reviewed the current diagnostic tests for Legionella and concluded that the PCR techniques offered the best hope of providing a highly sensitive and rapid diagnostic test for Legionella species infection.

Nocardia species

Nocardia is an aerobic filamentous bacterium belonging to the order Actinomycetales. Because Nocardia species exhibit many of the morphologic characteristics of fungi in culture, such as aerial hyphae, they have been classified in the past as fungi. Nocardia asteroides, the predominant pathogenic species, is recognized as a significant opportunistic pathogen in immunosuppressed patients. The lung is the usual site of introduction for Nocardia, where it can cause a wide spectrum of histologic injury from minimal infiltration to abscess formation and necrotizing pneumonitis. Disseminated disease can follow lung infection and can progress to abscess formation in the brain. Sinus tract formation is a characteristic trait of disseminated Nocardia infection, with tracts forming from the mediastinum or from satellite abscesses in subcutaneous tissue and skin from hematogenous spread. Nocardia is a relatively more common opportunistic pathogen in cardiac transplant recipients in particular, but is seen rarely in patients with AIDS for unknown reasons.

Diagnosis of this bacterial infection is based on demonstration of the organism in culture or by staining. Figure 14-5 illustrates a gram-stained section of material taken from a pulmonary abscess caused by N. asteroides. Because N. asteroides is not stained by hematoxylin and eosin or the periodic acid Schiff stain, it cannot be recognized unless Gram's stain or a methenamine silver stain is used. Although some stains of Nocardia may show an ability to retain an acid-fast or auramine stain, smears or sections have to be decolorized by a milder solution of acid, 1% sulfuric versus 3% hydrochloric. The ability to identify Nocardia in acid-fast and auramine-stained specimens varies considerably between different strains. Nocardia grows well on media for bacteria, fungi, and mycobacteria, and no special media are required for its recovery. Serologic assays are under development. Kjelstrom and Beaman (1993) developed a serologic panel to identify N. asteroides infection in a murine model, and Salinas-Carmona and colleagues (1993) developed a solid-phase ELISA to confirm the diagnosis of Nocardia brasiliensis in human mycetoma cases.

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MYCOBACTERIA

Before the discovery of streptomycin and other chemotherapeutic agents, the diagnosis of tuberculosis was made on the basis of the clinical picture, a radiograph of the chest compatible with that of tuberculosis, and the demonstration of acid-fast bacilli in the sputum. With the development of antituberculous drugs, however, it became necessary to isolate the organism so that drug susceptibility studies could be made and resistance could be detected. As more cultures were made, variant strains from the typical type of organism (i.e., M. tuberculosis) responsible for tuberculosis in humans were found. In 1954, Timpe and Runyon described 100 such stains, proposing a grouping for atypical organisms. At present, these organisms are referred to as mycobacteria other than tuberculosis, popularized by Woods and Washington (1987), or as nontuberculous mycobacteria, used by Chester and Winn (1986), Contreras (1988), Fournier (1988), and their colleagues, among others, including Heifets (1997). Woods and Washington (1987) suggested a clinically oriented classification scheme for mycobacteria that does not include Mycobacterium tuberculosis, bovis, and africanum (Table 14-7).

Specimen Collection in Pulmonary Tuberculosis

Sputum is the specimen most easily collected for use in making the diagnosis of pulmonary tuberculosis. A series of three to five early-morning specimens is recommended because experience shows that the number of bacilli shed varies from day to day in patients excreting low numbers of organisms. This variation is probably related to intermittent focal ulceration of the bronchial mucosa, releasing different numbers of tubercle bacilli in the bronchi over irregular periods. Krasnow and Wayne (1969) showed that specimens collected by heated aerosol or nebulization after the patient arises in the morning produce positive culture results after shorter incubation times and with fewer contaminants than do specimens collected over 24 hours. The 24-hour specimens yielded more positive culture results, although they required longer incubation times and were more likely to be contaminated. Both types of specimens are of value. Collection at bronchoscopy of secretions for culture is best done by using bronchial washings or bronchial lavage, but these specimens should be processed immediately if local anesthetics have been used to facilitate passage of the bronchoscope. Bronchial brushes, used in collecting specimens for cytology, provide good specimens for culture. Note that after bronchoscopy, recovery of mycobacteria increases in sputum specimens collected over the succeeding 24 to 48 hours. Early-morning gastric aspiration for organisms swallowed during the night is recommended only for infants or children or for those patients whose sputum cannot be obtained naturally or by heated aerosol. The recovery of saprophytic, nonpathogenic species of Mycobacterium from gastric aspirates can mislead the clinician until such time as identification of the organism is complete. In early stages of disseminated miliary tuberculosis, sputum specimens may not show the organism before invasion and ulceration of the bronchial tree. Demonstration or isolation of the organism in miliary tuberculosis may best be accomplished by liver biopsy, bone marrow aspiration, or, possibly, CSF examination. Lung biopsy frequently is helpful.

Mycobacterial Staining

Stained smears of the concentrated sputum should be made to search for the organism and to observe the numbers shed, an indication of the activity of the infection. Staining is the first step in making the diagnosis of mycobacterial disease. Sputum smear results are positive for mycobacteria in about 60% to 70% of specimens yielding positive culture results. Smears may be stained by one of the classic acid-fast techniques (e.g., Ziehl-Neelsen) or by a fluorochrome

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A possible second step in mycobacterial identification is a nucleic acid amplification test on the sputum. Currently, two tests have been cleared for clinical specimens: the Amplified Mycobacterium Tuberculosis Direct Test (MTD) (Gen-Probe, Inc., San Diego, CA, U.S.A.) and the AMPLICOR Mycobacterium Tuberculosis Test (Roche Diagnostic Systems, Inc., Indianapolis, IN, U.S.A.). According to Woods (2001), the performance of both tests is excellent, with a sensitivity of at least 95% and a specificity of 100% when testing smears that are positive for acid-fast bacilli. In contrast, when testing smear-negative specimens, the sensitivity varied from 45% to 53% and specificity varied from 96% to 99%, depending on which test was used. Gen-Probe subsequently enhanced the MTD test, and it is now approved for smear-negative specimens. The enhanced MTD has a sensitivity of 85.9% and a specificity of 97.8%. Molecular testing allows the physician to establish an early diagnosis, plan the patient's treatment, and identify other potentially infected patients.

Wang and Tay (1999) studied the use of molecular techniques to detect M. tuberculosis in clinical specimens. They evaluated the PCR-based COBAS AMPLICOR Mycobacterium tuberculosis Test; the Amplified M. tuberculosis Direct Test (MTD), which is based on TMA, and the ligase chain reaction based LCx test (LCx M. tuberculosis, Abbott Diagnostic Division, Abbott Park, IL, U.S.A.) and compared them to smear and culture results. Their analysis showed that the sensitivities of the LCx, COBAS AMPLICOR, and MTD were 100%, 96.1%, and 98.6%, respectively, with the specificities of 99.3%, 100%, and 99.4%, respectively.

Mycobacterial Cultures

Isolation of M. tuberculosis and other mycobacteria from sputum and other types of contaminated clinical specimens is facilitated by a digestion procedure to release mycobacteria from mucin, kill contaminating bacteria, and concentrate the number of mycobacteria to a smaller volume. Specimens from sterile sites do not have to be decontaminated.

Previously, the concentrated specimen would be inoculated to a minimum of two and preferably three different types of culture media. These would include one with an egg base (Lowenstein-Jensen), one with an agar base (Middlebrook 7H11 agar), and one to suppress nonmycobacterial organisms. Incubation of all media in 5% to 10% CO₂ results in an increased yield and rate of growth. In 1993, Tenover and colleagues recommended that both broth and solid media be used for cultures because the organisms could be recovered faster in broth. In current practice, the concentrated specimen is inoculated to Lowenstein-Jensen and a broth (Middlebrook 7H9), with the broth being incubated in an automated system. Such systems include the radiometric BACTEC 460, the nonradiometric, MGIT 960, BACTEC 9000 MB (Becton Dickinson Diagnostic Instruments, Sparks, MD, U.S.A.), MB/BacT system (Organon Teknika, Durham, NC, U.S.A.), and the ESP Culture System II (Trek Diagnostics, Inc., Westlake, OH, U.S.A).

In 1977, Middlebrook and associates described a broth culture medium (7H12) containing carbon 14 (¹⁴C) palmitic acid that could be used for the detection of the growth of M. tuberculosis. The method relies on the measurement, in an ion chamber system, of ¹⁴C-labeled CO₂ released during the metabolism of palmitic acid by mycobacteria. The drawbacks of this system were the need to dispose of the radioactivity properly and the constant puncturing of the diaphragm to sample the vial head space for ¹⁴C. Initial studies with the radiometric BACTEC system indicated that an inoculum of 200 viable units of M. tuberculosis could be detected in 12 to 14 days.

The current BACTEC 9000 MB system is a nonradiometric detection system. The specimen is inoculated into a BACTEC MYCO/F-Sputa culture vial, which contains a modified Middlebrook 7H9 broth with CO₂. BACTEC PANTA/F antibiotic supplement, which contains the antibiotics polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin, is usually added to the vial. The vials are incubated at 37 C and agitated once every 10 minutes. In the base of each vial is a fluorescent sensor, which consists of silicon rubber impregnated with a ruthenium metal complex. The metal complex responds to the concentration of oxygen in the culture medium. The initial large amount of oxygen in the media quenches the fluorescence. As the mycobacteria grow and the oxygen is depleted, the indicator fluoresces when it is exposed to ultraviolet light at 365 nm. A photo detector reads the fluorescence. The culture vials are continuously monitored; consequently, the vials must remain in the instrument for up to 56 days.

Damato and colleagues (1983) showed that 70% of smear-positive specimens are culture positive in the radiometric procedure within 14 days, with or without the addition of polymyxin B, amphotericin B, carbenicillin, and trimethoprim to the medium, compared with 21 days by the standard procedure. Similarly, Morgan and associates (1983) found that detection times for recovery of M. tuberculosis from smear-negative specimens with radiometric and conventional culture systems were 13.7 and 26.3 days, respectively. More recently, Pfyffer and colleagues (1997) compared the detection time for M. tuberculosis between solid media and the BACTEC 9000 MB system. The BACTEC 9000 MB detected the organism at 12.2 days for smear-positive specimens and 18.1 days for smear-negative

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Mycobacterial Identification

Conventional tests for the identification of mycobacteria were slow because they depended on isolating the organism. These tests include such things as optimal temperature for growth, rates of growth, pigment production, niacin accumulation, reduction of nitrates to nitrites, Tween 80 hydrolysis catalase activity, and a variety of other tests.

The complete identification of all mycobacterial isolates is mandatory because some organisms are known to cause disease and some are not associated with disease (see Table 14-7). Also, susceptibility tests must be performed to select the proper therapy. Species identification can usually be accompanied by determining relatively few characteristics. Although the incidence of M. tuberculosis has continued to decline, except for a temporary increase in incidence during the mid-1980s until 1990, the incidence of disease from nontuberculous mycobacteria is becoming more common.

Rapid Identification Tests

According to Woods (2002), the rapid identification tests include cell wall lipid analysis by high-pressure liquid chromatography (HPLC), nucleic acid probes, and the NAP test (para-nitro- -acetylamino- -hydroxypropiophenone). HPLC involves detecting the cell wall lipids and comparing them to known medically important mycobacterial species. The techniques of these chromatographic methods, as well as their pros and cons, have been succinctly presented by Heifets (1997).

The NAP test is used in conjunction with the radiometric BACTEC 460 system. NAP selectively inhibits the growth of M. tuberculosis. After a mycobacterium was isolated from the BACTEC 460, it was inoculated into a culture vial containing NAP. If the organism grew, it did not belong to the M. tuberculosis complex; rather, the organism belonged to the group of nontuberculous organisms. Today, the identification process begins with nucleic acid probes. AccuProbe (GenProbe, Inc., San Diego, CA, U.S.A.) is the only probe available for culture confirmation of Mycobacterium tuberculosis, kansasii, avium, intracellulare, andgordonae.

Polymerase Chain Reaction based Sequencing Identification

Soini and Musser (2001) described PCR-based sequencing as the gold standard for the identification of mycobacteria. The method consists of PCR amplification of mycobacterial DNA with genus-specific primers and sequencing the amplicons (amplified DNA segments). The organisms are identified by comparing the nucleotide sequence with the reference sequences. The most commonly used gene is the 16S ribosomal RNA (rRNA) gene. This gene has both conserved and variable regions that make it an ideal target. The MicroSeq 5000 system (PE Applied Biosystems, Foster City, CA) sequences a part of the 16S rRNA gene. It can identify most mycobacteria in 2 days. Early experience indicates that the instrument identifies organisms more rapidly and accurately. Drawbacks of the system include expense and the inability to differentiate certain mycobacteria.

Mycobacterial Susceptibility Testing

With the advent of antibiotics to mycobacteria, the organisms had to be tested for susceptibility. The most common drugs tested are rifampin, isoniazid, ethambutol, and pyrazinamide. With older techniques, the organisms took between 2 and 6 weeks to isolate. The susceptibility testing could then take an additional 2 to 4 weeks.

Conventional Mycobacterial Susceptibility Testing

The first susceptibility testing was performed in plastic Petri dishes that were divided into four quadrants with Middlebrook 7H10 agar serving as the media. Antibiotics were incorporated into the agar in three of the four quadrants, with the fourth quadrant serving as a growth control. Each quadrant was inoculated with between 100 and 300 organisms. A second set of identical plates was inoculated with a 100-fold dilution. The object of this dilution was to determine whether 1% of the organisms were resistant to the antibiotic. If 1% of the organisms are resistant, then the antibiotic is not clinically useful.

When the broth culture system came into use, it decreased the time for susceptibility testing. The radiometric BACTEC 460 was used for susceptibility testing. Snider and colleagues (1981), in a multicenter collaborative study, reported that drug susceptibility tests of M. tuberculosis with radiometric and standard methods were similar. They also found that agreement between the BACTEC and agar dilution was better when comparing drug-resistant strains. Siddiqi and associates (1981) found that 98% broth susceptibility results were reportable in 5 days and that the agreement between radiometric and agar dilution susceptibility testing was 95%.

According to Woods (2002), only the radiometric BACTEC 460 system and the newer ESP Culture System II

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Rapid Mycobacterial Susceptibility Testing

Molecular testing for susceptibility is faster than the current conventional testing. To perform molecular testing, one must understand the genetic basis for drug resistance. Ramaswamy and Musser (1998) pointed out that the genetic basis for rifampin resistance is simple and well characterized, whereas the molecular basis for the resistance to other drugs is much more complex. Rifampin binds to the -subunit of RNA polymerase, which is encoded for by the rpo B gene, and inhibits transcription initiation. Almost all rifampin-resistant isolates have point mutations in an 81 base pair region of this gene. The various point mutations stop rifampin from binding to the -subunit of the RNA polymerase. These mutations are absent in susceptible organisms. Isoniazid (INH) resistance is more complex because it involves multiple genes. However, mutations in two genes, kat G and inh A, are found in 75% to 85% of INH-resistant tuberculosis.

Soini and Musser (2001) described PCR-based sequencing, the line probe assay, and DNA microarrays as being able to detect mutations in such genes as rpo B. PCR-based sequencing involves sequencing the rpo B gene and comparing it to the normal sequence. If the gene shows a mutation, the organism is most likely resistant to rifampin. As the molecular biology of mycobacterial drug resistance is understood, examining the genetic structure by some of the aforementioned techniques will yield a rapid result as to whether or not the organism is susceptible or resistant to an antibiotic.

FUNGAL INFECTION OF THE LUNG

The spectrum of fungal infection of the lung is shifting from the endemic, deep-seated infection, such as blastomycosis, to opportunistic infection with usually low pathogenic species, such as Candida and Aspergillus species. Fungal diseases are diagnosed in much the same way as mycobacterial diseases: visualization, cultures, molecular techniques, and clinical laboratory tests, that is, serology and antigen detection.

Fungal Staining

Pulmonary fungal infections are best established by the recovery of the infecting organism in a culture. Morphologic changes in tissue biopsy specimens may be adequate to establish a diagnosis without culture. The first step in identifying a fungus is microscopic examination of the tissue. Figures 14-6, 14-7, 14-8, and 14-9, respectively, show a silver stain of the organisms of histoplasmosis, blastomycosis, coccidioidomycosis, and cryptococcosis. Histochemical staining, with periodic acid Schiff, methenamine silver, mucicarmine, or Gram's stain, of histologic sections is helpful and may afford specific identification of different fungi. Monteagudo and associates (1995) have described an immunohistochemical staining method using a specific monoclonal antibody to identify Candida albicans. Moskowitz and colleagues (1986) also used immunohistochemical staining to identify systemic fungal infections. Perhaps the two best stains for demonstration of fungi in tissue are periodic

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Fungal Cultures

Cultures of fungi can be made from tissues, sputum, pleural fluid, bronchial aspirates, or other clinical specimens. For optimal recovery, all specimens should be inoculated on several different types of culture media. Sabouraud's dextrose agar is an excellent general-purpose culture medium. It is able to inhibit many strains of contaminating bacteria because its high dextrose content (4%) reduces the pH to 5.6. Specimens also should be inoculated to a second medium containing antibiotics and cycloheximide to suppress less fastidious bacteria and the contaminating molds. Because the cycloheximide and antibiotics in the second medium also inhibit certain pathogenic fungi, such as Cryptococcus neoformans and Aspergillus fumigatus, use of Sabouraud's or a similar noninhibitory agar should not be omitted. Sabouraud's medium should be incubated at both 25 C and 37 C because different fungi may have varying rates of growth and different morphologic forms when grown at different temperatures. Fungi with more than one form are dimorphic, showing a yeastlike morphology at 37 C and a mycelial growth when incubated at room temperature (i.e., 25 C). Examples of dimorphic fungi are Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis. Growth of pathogenic fungi may take from 2 to 14 days, depending on the number of organisms present in the specimens and characteristics of the individual organism. For some fungi, unique growth requirements have led to special media. Any clinical information that may indicate the most likely organism helps in selecting the medium most likely to produce growth in the minimal time.

Members of the Candida and Cryptococcus species are easily identified by fermentation and carbohydrate assimilation tests. The use of several other biochemical tests in the speciation of fungi is helpful, but most pathogenic fungi are identified by a variety of ways that include appearance of the colony, rate of growth, colony pigmentation, growth on media containing antifungal agents, and dimorphic growth. Microscopic examination involves taking some of the filamentous mold from the colony and mounting it on a glass slide with a lactophenol aniline blue stain. The slide is examined under the microscope for such features as septate or nonseptate mycelia, unique macroconidia, or microconidia.

Molecular Techniques for Detecting Fungus

Some fungal organisms may grow slowly, but they are easier to identify under the microscope because they are larger than bacteria and have a distinct morphology. Saubolle (2000), in his excellent review of fungal pneumonias, mentioned that nucleic acid probes are available for the rapid identification of Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis. These probes allow accurate identification of an organism shortly after it is isolated (AccuProbe). Chemaly and colleagues (2001) took the H. capsulatum AccuProbe to the next level and used it on a clinical specimen. They described a 58-year-old man who had an H. capsulatum endocarditis. They tested the specimen with the AccuProbe, and it was positive for H. capsulatum. A week later, the culture grew a fungus that was identified by AccuProbe as H. capsulatum.

Fungal Serology and Antigen Detection Tests

Many patients have had subclinical infections with different fungi; hence, a positive reaction to a serologic test for a

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Saubolle and McKellar (2001) describe serology as valuable in diagnosing H. capsulatum and C. immitis, but not B. dermatitidis, C. neoformans, P. carinii, or other fungi. Martynowicz and Prakash (2002) attempted to diagnose 25 patients with known blastomycosis by serologic tests that included immunodiffusion and complement fixation. The immunodiffusion was positive in 10 of 25 patients (40%), and complement fixation was positive in 4 of 25 patients (16%). These results again confirm that serology is not useful for the diagnosis of blastomycosis.

Wheat (2001) described the serologic test for histoplasmosis as being sensitive, and if properly performed, the tests are positive in 90% of patients. The testing has some limitations, just as it does for most infections. First, the antibody response takes 2 to 6 weeks. Second, immunosuppressed patients may not have a serologic response. Third, a positive serologic response may be from an old infection. The two standard tests for measuring antibody response to H. capsulatum are immunodiffusion and complement fixation. Immunodiffusion is simple to perform and more specific than complement fixation but is less sensitive when used for screening. Immunodiffusion detects the H (IgM) and M (IgG) precipitin bands. M precipitins are detected in 75% of patients with acute histoplasmosis and nearly all patients with chronic histoplasmosis. Fewer than 20% of patients show H bands. The H precipitins disappear after 6 months, but the M precipitins may persist for years. The complement fixation test is positive in 95% of patients infected with histoplasmosis, but one fourth of those patients are weakly positive, with titers of 1:8 or 1:16. Titers in this range may indicate a previous infection, or the patient may have active disease with a weak response. Skin testing for histoplasmosis may cause the titers to rise and give the impression of an active infection.

Wheat and colleagues (2002) believe that testing for a histoplasma-specific glycoprotein in urine, serum, bronchoalveolar lavage fluid, and CSF is an important adjunct in diagnosing histoplasmosis. The antigen test is the form of an enzyme immunoassay and is only offered from the Histoplasmosis Reference Laboratory at Indiana University (1001 W Tenth St., Indianapolis, IN 46202; 1 800-HIS-TODG). The specificity for the test is 98% because there can be some false-positive reactions. The sensitivity is 90% in patients with disseminated disease. The urine is more frequently positive (92%) than the serum (82%). The sensitivity in patients with acute pulmonary histoplasmosis is 75%.

Stevens (2002) described the serology of coccidioidomycosis as one of the best tests ever produced for fungal infection. Enzyme immunoassays are available for IgM and IgG antibodies to the fungus. The IgM antibody appears early, indicating an acute infection. Over several months, the IgG appears and increases in strength. The serology is useful because it inversely follows the response to therapy. For example, if the patient is under treatment and the antibody titer is dropping, the patient is responding to treatment.

C. neoformans usually enters the body through the lungs. The yeast may remain in the lungs, or it may disseminate to other parts of the body, particularly the central nervous system, skin, bone, and lungs. Dissemination is facilitated by an immunosuppressed state, such as AIDS or cancer chemotherapy. Saubolle and McKellar (2001) described latex agglutination and ELISA systems that are available to detect cryptococcal polysaccharide in the serum or CSF during dissemination or meningeal disease. The tests have a high sensitivity (95%) and specificity (98%) in the disseminated form of the disease. However, when the infection is limited to the lungs, the sensitivity for the serum antigen detection drops to 50%.

Pneumocystis Pneumonia

Schliep and Yarrish (1999) reviewed the subject of P. carinii pneumonia infection. The organism was recognized simultaneously by Carlos Chagas and Antonio Carini, after whom it was later named, in the early 1900s. The organisms were first described in rodents. The infection generally occurs in immunocompromised patients. Stringer and colleagues (2002) point out that the organism was originally thought to be a protozoan, but in 1988, DNA analysis demonstrated that Pneumocystis is a fungus. In 1999, the organism that causes human Pneumocystis infection was renamed P. jiroveci. Stringer and associates (2002) also believed that the medical literature could retain the acronym PCP, because it could be read Pneumocystis pneumonia.

P. jiroveci (Fig. 14-10) is a frequent complication in patients with AIDS. Attempts to isolate and culture the organism have been unsuccessful. The disease was first recognized in malnourished infants and children in orphanages in Europe and Korea after World War II. Yale and Limper (1996) at the Mayo Clinic reviewed the occurrence of P. jiroveci pneumonia in 116 patients without AIDS. They found that 30.2% had hematologic malignancies, 25% had undergone organ transplantation, 22.4% had an underlying inflammatory disease

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The organisms of Pneumocystis still cannot be grown in culture. The diagnosis is established by demonstrating the presence of cysts in tissues or respiratory secretion, such as an induced sputum or BAL. The wall of the cyst stains with Gomori's methenamine silver, Gram-Weigert, and toluidine blue O. Amin and associates (1992) and Blumenfeld and Kovacs (1988) described the immunostaining of the cysts. Immunofluorescent stains are also performed on the cysts. Molecular techniques have also been developed for the diagnosis of Pneumocystis. Kasolo and co-workers (2002) used PCR to detect P. jiroveci in the lung tissue of children who died. They found a sensitivity of 100% and a specificity 63%. They concluded that the low specificity precludes the use of PCR as an alternative to microscopic examination. Figure 14-10 is a silver stain showing the typical cysts.

PARASITIC LUNG INFECTIONS

Pulmonary parasitic infections are uncommon in the United States. They are usually found in returning travelers who have been to parts of the world where parasitic infections are common or in immunosuppressed patients who had a low-grade parasitic infection before their immunosuppression. Parasites are difficult to recover in sputum, pleural fluid, BAL fluid, or some other clinical specimen. Savani and Sharma (2002) described the following parasites as involving the lung: Strongyloides stercoralis, Paragonimus westermani, Echinococcus granulosus, Entamoeba histolytica, schistosomiasis, and filarial infections. Other parasites that can involve the lung include malaria, hookworm, and Ascaris lumbricoides. Most of these parasitic infections are diagnosed by examining the appropriate clinical specimen, usually stool, for the eggs of the parasite.

Strongyloides stercoralis

Wehner and Kirsch (1997) reviewed the pulmonary manifestation of strongyloidiasis. The organisms are endemic worldwide in tropical and subtropical areas as well as in the southeastern United States. The filariform larvae penetrate the skin and migrate hematogenously to the lung, where they ascend the airway and are swallowed. They mature in the gut. Immunosuppressed patients may develop overwhelming infection that can involve the lung, and the larvae can be seen in the sputum. A diagnosis is established by finding the larvae in stool, body fluids, or tissues.

Paragonimus westermani

P. westermani, the lung fluke, is endemic in Korea, Japan, China, and other parts of Southeast Asia. In the United States, the patient should come from one of these areas and have a history of eating raw crab or crayfish. Nakamura-Uchiyoma and colleagues (2002) described the patients as having an elevated IgE and an eosinophilia. The diagnosis is established by finding the eggs in the stool or the sputum. Clinical laboratory tests, using ELISA, detect parasite-specific IgG or IgM antibodies.

Echinococcus granulosus or Pulmonary Hydatid Disease

E. granulosus is a small cestode tapeworm that lives in dogs. The eggs shed in their feces are infective for intermediate hosts, including humans. The egg releases an oncosphere that, when ingested by a human, usually lodges in the lung or liver. In the organ, the oncosphere matures into a hydatid cyst. Gottstein and Reichen (2002) pointed out that imaging studies and serology establish the diagnosis. The serologic tests are aimed at detecting serum antibodies or circulating antigens. The indirect hemagglutination tests and ELISA using hydatid fluid antigen are positive in 85% to 98% of hepatic cases but in only 50% to 60% of pulmonary cases. The antigen-5-precipitation test (arc-5-test) and immunoblotting for the 8KD/12KD hydatid fluid antigen are more specific tests.

Entamoeba histolytica

E. histolytica is usually a protozoan infection of the gastrointestinal tract. The organism can spread and produce abscesses in the brain, liver, and lungs. Shamsuzzaman and Hashigurchi (2002)

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Serologic tests exist that can detect antibodies to E. histolytica in serum and in pus. Some qualifications exist in diagnosing amebiasis by IgG antibodies. First, the antibodies may not be present at an early stage of the disease. Second, antibodies remain elevated after treatment. Finally, antibodies may develop in cases with asymptomatic colonization of the intestine. The detection of IgM antibodies may be more useful because they appear shortly after an infection and only persist for a short time. Abd-Alla and colleagues (1993) described detecting the galactose-inhibitable adherence protein (GIAP) of E. histolytica in the serum as a means of diagnosing the infection. Subsequently, Abd-Alla and associates (1998) described detecting the serum IgM antibodies to GIAP as useful in diagnosing liver abscesses. In summary, serologic tests are useful in suspecting the diagnosis, but identification of the organism is still important.

Schistosomiasis

Schistosomiasis is one of the most prevalent infections in the world, with an estimated 200 million people being infected. The parasites exist in Africa, China, Japan, South East Asia, South America, and the Caribbean. Schistosomes are a group of trematodes (flukes) that live in the blood vessels of their host. Schwartz (2002) divided pulmonary schistosomiasis into an acute (early) disease, usually involving travelers returning from an endemic area, and a late disease with the complications that occur in an endemic area. Early disease occurs 3 to 8 weeks after infection, whereas chronic disease occurs after being infected for many years and the eggs are deposited in the pulmonary vasculature (Fig. 14-11). In early disease, the imaging studies may be helpful. As for laboratory studies, microscopic examination of the stool and urine has a low sensitivity in early disease, but in late disease, eggs should be present. The eggs may also be seen in bladder or rectal biopsies.

Serologic methods are directed at detecting specific antibodies or circulating antigens. Ross and colleagues (2002) described antibody detection as being useful under some circumstances. Its usefulness is limited because the antibodies persist following successful treatment. Detection of circulating antigen from the adult worms or eggs has been shown to be a useful test by Wang and co-workers (1999). After treatment, the circulating antigens would be expected to decrease.

Other Parasites

The lung can also be involved with malaria, hookworms, ascariasis and filariasis. Taylor and White (2002) described nonhydrostatic and noncardiogenic pulmonary edema as the most significant malaria-induced injury. The condition is well described with Plasmodium falciparum, but may also occur with Plasmodium vivax and ovale. Examination of both thick and thin peripheral blood smears leads to the correct diagnosis. With filariasis, antibodies to the parasites can be detected in the blood. Ascaris and hookworm may incite a severe inflammatory reaction in the lung during passage from the pulmonary circulation into the bronchi. Sputum specimens may reveal the filarial forms of the worms. Examination of stool specimens usually shows the eggs of both organisms.

VIRAL INFECTIONS OF THE LUNG

Although viral infections of the upper and lower respiratory tract are among the most frequent illnesses in humans, most of these infections are benign and self-limited. The most common etiologic agents are influenza virus, parainfluenza virus, adenoviruses, and respiratory syncytial virus. The situation is different in the immunosuppressed patient, in whom any of these agents can cause serious life-threatening infection. Viral infections of the lung account for most opportunistic viral infections in immunosuppressed patients. In patients with AIDS, Klatt and Shibata (1988) found that cytomegalovirus (CMV) pneumonitis was present at autopsy in about 30% of patients, and evidence of the virus was present in most lung tissue examined. CMV is rarely the cause of death in these patients, however, but the

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HERPESVIRUSES

The family of herpesviruses is unique because most of the general population is exposed to them and carries the virus in a latent form. The family contains such members as CMV, EBV, VZV, HSV types 1 and 2, and human herpesvirus (HHV) types 6, 7, and 8. In the latent state, the virus lies dormant in a virus-specific privileged area within the body (i.e., lymphocytes for EBV and nerve ganglia for VZV). In the immunocompromised patient, reactivation of the virus or a primary infection can lead to serious opportunistic infection present as overwhelming disseminated disease. CMV and EBV are also strongly immunomodulating, particularly in immunosuppressed individuals. These features of herpesviruses make them fascinating and a particular challenge in the immunosuppressed patient.

The demonstration that ganciclovir is an effective agent in the treatment of CMV infection in immunosuppressed patients by the Collaborative DHPG Study Group (1986) and others has stressed the importance of early and accurate diagnosis. Whether infection in the transplant recipient represents primary or secondary infection is of great importance in patients who undergo organ transplantation because mortality from primary infection is far greater when compared with reactivation of latent infection. Primary infection in these patients usually represents receipt of an organ from a CMV-positive donor or blood products contaminated with the virus. Thus, serologic status is crucial in the pretransplantation evaluation of these patients. Demonstration of IgM class antibodies or an increase in antibody titers is confirmatory evidence of reactivation, but these events occur relatively late in the course of illness and are not generally helpful in the diagnosis of acute disease.

Herpes Simplex

Whitley and Roizman (2001) reviewed HSV types 1 and 2 infections. HSV-1 is associated with orofacial infections and encephalitis. HSV-2 causes genital infections and may be transmitted from infected mothers to their babies. HSV may be associated with pneumonia in patients who have received transplants. Taplitz and Jordan (2002) point out that, histologically, the virus causes necrosis, hemorrhage, and a mononuclear infiltrate. The infected epithelial cells have a typical gray to eosinophilic nuclear inclusion. HSV can be detected in a cell culture in about 24 to 48 hours. The identity is confirmed by immunofluorescence or enzyme immunoassay. PCR may be performed on BAL fluid.

Cytomegalovirus

Early detection of CMV can be done in a variety of ways. A tissue biopsy or a cytology specimen shows the typical intranuclear inclusion that is described as an owl's eye (Fig. 14-12). Both immunostaining and ISH can be performed on tissue specimens. Boeckh and Boivin (1998) reviewed the methods for detecting CMV. They include cultures, detection of CMV antigens in the blood (pp65), PCR assay, branched DNA signal amplification assay, and hybrid CMV DNA assay. Taplitz and Jordan (2002) pointed out that these new methods are very useful in managing patients. They do have some drawbacks, however, because they cannot always distinguish between active and latent infection. The CMV antigenemia assay requires the isolation of neutrophils, which are then stained with a monoclonal antibody to pp65. The reaction is visualized through immunoperoxidase staining or immunofluorescence. The sensitivity is high (up to 95%), but the specificity is lower (80% to 90%).

Epstein-Barr Virus

Cohen (2000), in a review of the EBV, pointed out that 90% of the world's population is infected by the virus and that the infection persists for the lifetime of the patient. In younger patients, the virus causes infectious mononucleosis with its triad of fever, lymphadenopathy, and pharyngitis. EBV does not usually cause pneumonitis. In transplant recipients, it may involve the lung through a posttransplantation lymphoproliferative disorder (PTLD). According to Taplitz and Jordan (2002), ISH can detect EBV RNA in infected

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Varicella-Zoster Virus

Lung involvement with primary VZV is not common in immunocompetent patients. In immunosuppressed individuals, however, primary VZV infection and pneumonia can be life threatening. Reactivation of VZV (i.e., shingles) in recipients of organ transplants also can progress to invasive pulmonary disease. The diagnosis of VZV is usually not difficult because of the characteristic exanthem in primary cases and the presence of shingles in reactivation cases. Serology, however, does play an important role, especially in transplant recipients, because pretransplantation seronegativity should lead to administration of soon-to-be-released vaccine. In addition, postexposure varicella-zoster immune globulin can be administered and is effective in decreasing morbidity in immunocompromised patients, as demonstrated by Zaia and co-workers (1983). Serum titers for VZV to confirm seroconversion or the presence of IgM are done. It is important to note that cross-reactivity is substantial, up to 33%, between antibody assays for HSV and VZV, and the absence of a similar titer increase to HSV may be necessary in these situations. In case of diagnostic uncertainty, materials from lesions or biopsy specimens can be submitted for routine culture, shell vial culture, and immunofluorescence staining, the latter being the most efficient method.

Human Herpesvirus

The most important HHVs are serotypes 6, 7 and 8. Caserta and colleagues (2001) reviewed HHV-6 and pointed out that it is associated with febrile illnesses and specifically with roseola infantum in children within the first 2 years of life. HHV-7 has been associated with a roseola-like illness in young children. Allen (2002) describes HHV-8 as being associated with Kaposi's sarcoma, primary effusion lymphoma, and a form of Castleman's disease. As far as pneumonias, Taplitz and Jordan (2002) do not believe HHV-7 plays a role in pneumonia following hematopoietic cell transplantation. They are not certain about the role of HHV-6 or -8, but HHV-8 related Kaposi's sarcoma has been reported in immunosuppressed patients. Dockrell (2003) indicated that HHV-6 has been associated with pneumonitis following bone marrow transplantation. HHV-6 can be cultured from specimens with the shell vial technique reducing the culture time. Serologic tests are available with the presence of HHV-6 IgM or a fourfold rise in IgG supporting a clinical diagnosis.

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