Diagnosis of community-acquired pneumonia
The diagnostic approach to patients with CAP includes, clinical evaluation, followed by radiographic and microbiologic tests. Most studies from United States as well as other parts of the world indicate that a microbiologic diagnosis in CAP is only confirmed in 50-70% of cases. Even using invasive techniques such as lung and tracheal aspirates yield the etiology in up to 65% of those who were negative with conventional techniques of sputum culture. Microbiologic tests include sputum smear, sputum culture and blood culture in all patients suspected of having pneumonia.

Sputum examination may not yield the etiologic agent, since about 30% of patients cannot produce optimum sputum sample, and approximately 25% of patients have already received antibiotics. In spite of these drawbacks, effort should be made to identify the causative agent in order to optimize therapy.

Gram stain of sputum smears has been valuable in determining the etiologic bacteria in experienced hands, using the following guidelines:

1. If there are > 25 neutrophils and < 10 epithelial cells / low power field, the sputum is considered a purulent sputum.
2. If a predominant type of bacterium is found in the smear, it may be the cause of pneumonia.

Pathology of Bacterial Pneumonias
The pathology of bacterial pneumonias may be discussed as related to the three groups of patient populations:

Community Acquired Pneumonias Pneumonias in hospitalized patients Pneumonias in HIV-infected patients

Since several bacteria are common in each group of these patients, in this presentation the pathologic findings are discussed in two groups based on the occurrence of infection, as commonly encountered pneumonias and uncommon/emerging pneumonias.

Common Bacterial Pneumonias

Streptococcus pneumoniae: This is the most commonly encountered infection in all groups of patients, especially in infants and in adults >60 years of age, and accounts for approximately 66% of all bacteremic CAP. The incidence of S. pneumoniae infection has increased 2-3 folds in the past 20 years, particularly in HIV- infected patients and cigarette smokers. S. pneumoniae are gram positive, fastidious and fragile alpha-hemolytic bacteria that grow best in 5% CO2, and require a source of catalase to grow on agar plates. The bacteria are spherical or ovoid, measure 0.6-1.0 um in diameter, and have a capsular polysaccharide as the major protective antigen. Streptococci use cell wall proteins, particularly choline to attach to human cells using the human cell carbohydrates or the platelet activating factor (PAF) receptor. Once attached to the cell wall PAF, the bacteria enter the host cytoplasm in an endocytic vacuole, and then transported across the cell into adjacent tissues. The damaging effects of pneumococci are mediated through production of hemolysins, nitric oxide and interleukin-1, and induction of neutrophil influx through ICAM-1.

Pathology: S. pneumoniae has been the most common cause of lobar pneumonia. Descriptions of the stages of evolution of the pneumonia are found in reports of the early twentieth century casualties, before the discovery of penicillin. The gross and microscopic findings in the lungs depend on the stage of infection. The four stages of lobar pneumonia as classically described in untreated patients in the literature are rarely seen now. During the acute early phase, grossly, there is edema with consolidation often in a lobar pattern, with marked alveolar and interstitial edema and alveolar capillary congestion. Gram stain reveals abundant gram-positive cocci in the edema fluid. This stage is followed by the stage of red hepatization and then gray hepatization. Red hepatization stage shows lobar consolidation with congestion. Histologically, there is abundant fibrinous exudates associated with congestion. Gray hepatization stage is characterized by tan gray color of the consolidated areas, with initially an exuberant alveolar neutrophil infiltrate followed by macrophages. The last stage of resolution is characterized by the presence of macrophages and removal of the debris. There is associated pleuritis and pleural effusion in severe infections. Since there is no destruction of the alveolar septa, no permanent damage is seen in the lungs following recovery.

Mycoplasma pneumoniae:
Mycoplasma are the smallest free-living organisms that cause disease in humans. These are filamentous, pleomorphic organisms that lack a cell wall, divide by binary fission, and measure 200 x 10 nanometers. The infection is transmitted from person-to-person by droplets, particularly in fall and winter; with highest infection rates in school children, military recruits and college students. Incubation period is approximately 3 weeks, with a slow onset of symptoms, although many infections are asymptomatic. Extrapulmonary symptoms are often associated with the respiratory symptoms, and include hemolysis, skin rash, and joint pains. Hemolysis is due to the appearance of I antigen on erythrocyte membranes with production of a cold agglutinin response; the diagnostic titers range from > 1:128 to 1:50,000. Chest x-rays most commonly show a peribronchial pneumonia pattern with atelectasis in the lower lobes, and less commonly nodular infiltrates and hilar lymphadenopathy.

Once inhaled, the organism attaches to the epithelium using adherence proteins, and produces hydrogen peroxide and superoxide radicals, resulting in epithelial cell injury. In addition, an immune mediated injury causes the pathologic changes.

Isolation of M. pneumoniae is difficult but possible using SP-4 medium since the organism is fastidious, and requires 2-3 weeks for growth. The Complement Fixation (CF) test is highly sensitive for diagnosis, however, false positives may occur. Positive results = > 4 fold increase in paired sera or a single titer > 1:32. Other diagnostic tests include ELISA, antigen capture-enzyme immunoassay (Ag- EIA) and PCR; the latter detects genomic DNA and may be highly sensitive and specific.

Pathology: The lungs show acute and chronic bronchiolitis and peribronchiolitis which extends in to the adjacent lung parenchyma. There is necrosis and sloughing of airway epithelium with neutrophil infiltrate within the bronchioles and lymphoplasmacytic infiltrate in the bronchiolar walls. The adjacent lung parenchyma may show Type II pneumocyte hyperplasia. Fatal cases may also have diffuse alveolar damage.

Haemophilus influenzae:
Hemophilus influenzae are small, non-motile, gram-negative coccobacilli or pleomorphic bacilli that grow on enriched media such as chocolate agar. H. influenzae is a common cause of mild and uncomplicated upper respiratory infections. Rarely, severe epiglottitis, bacterial meningitis, pneumonia, and bone and joint infections may be seen in patients with chronic pulmonary diseases and alcoholism. The pathogenecity of the organism is determined by the presence of a polysaccharide capsule, with type B and E strain being frequent pathogenic isolates.

Pathology: The inflammatory response is primarily neutrophils, producing a bronchopneumonia with purulent exudate. Necrotizing bronchiolitis and abscesses may be also seen. Gram stain lacks sensitivity since the bacteria are very small and obscured by fibrinous exudates. Smears stained with acridine orange, and examined under a fluorescent microscope have a better diagnostic yield.

Staphylococcus
Staphylococci are gram-positive cocci, measuring 0.7-1.2 um in diameter, with a tendency to grow in clusters. The bacteria produce a large number of enzymes and toxins including catalase, coagulase, hyaluronidase, and beta-lactamase. Staphylococci colonize the skin, anterior nasal cavity and sometimes the gastrointestinal tract, resulting in 20-40 % of the population becoming a carrier. Staphylococci are an important cause of post-influenza bacterial pneumonia in the chronically ill and debilitated population. Staphylococcal pneumonia may result from inhalation or aspiration of oropharyngeal secretions, and from hematogenous spread from another infected site such as endocarditis or skin abscesses etc. The characteristic feature of infection is early necrosis and abscess formation, with extension of inflammation in to the pleura resulting in an empyema.

Pathology: Histologically, in the acute stage of infection, there is suppurative bronchitis and bronchiolitis with or without pseudomembranes, pulmonary edema and hemorrhages. At a later stage, areas of consolidation with central necrosis develop, with formation of abscesses. The abscesses contain clusters of hematoxyphil staining, gram-positive bacteria. The bacterial colonies may be surrounded by an acellular eosinophilic matrix rich in immunoglobulins, (Splendore-Hoeppli phenomena), also called "botryomycotic abscess".

Pseudomonas
P. aeruginosa is an opportunistic infectious agent, ubiquitous in the environment, particularly in warm water reservoirs. Nosocomial infections with P.aeruginosa are most common in the intensive care units, in surgical and burn units, and in patients with cystic fibrosis. Clinical presentation of patients with Pseudomonas pneumonia is often fulminant, with severe dyspnea, cyanosis, gram-negative sepsis and respiratory failure. The bacterium is aerobic, non-spore forming gram-negative rod, measuring 1.0-5.0 um in length and 0.5-1.0 um in width, with polar flagella. Most isolates grow on agar plates, producing a distinctive musty, grape-like smell.

Pathology: Pseudomonas bronchopneumonia is commonly characterized by necrosis, hemorrhage, and large numbers of gram-negative bacilli, in a predominantly vasocentric pattern, with minimal or absent neutrophil infiltrate. In some cases, the inflammation and necrosis are centered on the airways, producing a necrotizing bronchopneumonia with abscesses. The latter pattern is common in cystic fibrosis patients, who often have a long chronic course with multiple bouts of pneumonia and bronchitis, ultimately developing bronchiectasis. Brown-Hopps stain is superior to Brown-Brenn for demonstration of the bacteria in tissue sections.

Uncommon/Emerging Bacterial Pneumonias

Actinomyces
Actinomyces are anerobic or microaerophilic filamentous bacteria, commensal in the oropharynx, vagina and gastrointestinal tract of healthy people that cause endogenous infection. There is no increased incidence of infection in immunocompromised individuals. Thoracic actinomycosis is caused by aspiration of oropharyngeal contents.

Pathology: The characteristic histological lesion is an abscess with one or more actinomycotic granules in the center of the abscess. The granules consist of aggregates of delicate, filamentous, branching, amphophilic, often beaded bacteria, measuring 1.0 um in width. At the periphery of the granules the filaments are radially oriented and covered with finger-like, deeply eosinophilic Splendore- Hoeppli material. The granules are surrounded by neutrophils, and represent the "sulfur granules" seen in the draining actinomycotic sinuses. The bacteria are gram-positive (Brown-Brenn), acid-fast negative, and stain with silver stains. Gram-stained smears of the sulfur granules show the filamentous bacteria with right-angled branching pattern, and beading. Culture and immunoflourescent antibody may be used for confirmation of diagnosis.

Nocardia
Nocardiae are aerobic bacteria resulting in opportunistic infection in immunocompromised hosts. Nocardiae are distributed world wide as saprophytes in the soil and decaying vegetable matter. Inhalation of the bacteria causes pulmonary nocardiosis with formation of abscesses, cavitary lesions and suppurative consolidation. The lower lobes of the lung are frequently involved with development of suppurative pleuritis and empyema.

Pathology: The suppurative abscesses contain delicate, gram-positive, randomly oriented filaments with right-angled branching pattern. The bacteria measure 1.0 um in width and 10-50 um in length, and have a characteristic beaded pattern due to irregular gram staining. The Splendore- Hoeppli material is not seen in nocardia abscesses. Similar to actinomyces, these bacteria stain positive with gram stain (Brown-Brenn and Brown-Hopps), silver stain (GMS), but often not with PAS stain. Unlike actinomyces, nocardia are partially acid-fast and non-alcohol fast with Kinyoun, Coates-Fite and Fite-Farraco stains. Nocardia infections are often difficult to diagnose in routine H & E stained sections, and often take 1-2 weeks for culture.

Rhodococcus
Although known to veterinarians for a long time, human disease from rhodococcus was first reported in 1967, with a few more cases reported since then. The bacteria are widely distributed in soil and animal feces, and inhalation of contaminated dust is the most likely source of infection. The infections are almost exclusively seen in immunocompromised patients, and more commonly in men.

Pathology: Rhodococcus equi is a facultative intracellular, aerobic, gram-positive, non-motile, partially acid-fast, pleomorphic cocobacillus measuring <1.0 um in diameter. The lesions are grossly seen as areas of consolidation with central necrosis and cavitation. Histologically, a granulomatous infiltrate is seen, consisting predominantly of sheets of macrophages mixed with variable number of neutrophils, plasma cells and lymphocytes. The macrophage cytoplasm has PAS positive granular material that is shown to be giant phagolysosomes containing degenerated bacteria and cellular debris. In addition, small round, target-shaped, calcified cytoplasmic inclusions may also be present, the Michaelis-Gutmann bodies (MG). The granulomatous inflammation with MG bodies may present as a nodular pulmonary mass or malakoplakia, with histologic features similar to malakoplakia in other body sites. Special stains, Gomori's methenamine silver, Grocott, Giemsa, von-Kossa, Alizarin red and Prussian blue help in identification of the MG bodies. The R. equi stain gram-positive with Brown-Brenn or Brown-Hopps, black with silver stains, and partially acid-fast with modified acid-fast stains such as Fite-Farraco, or modified Kinyoun, but not with Ziehl-Neelson stain.

Legionella
Legionella pneumophilia, are aerobic, gram-negative bacilli, with a ubiquitous aquatic distribution, particularly in water reservoirs. The organism was first described by McDade in 1977, and is now known to cause both sporadic and epidemic infections. The pulmonary infection is often associated with symptoms of gastrointestinal, cerebral involvement and often serum electrolyte abnormalities. The bacilli are 0.3-1.0um wide and 2.5 um long, and are facultative intracellular organisms that proliferate in phagocytic cells.

Pathology: The lungs are consolidated, firm and have focal necrosis. The alveoli are filled with fibrin, and sheets of macrophages and neutrophils. Progression of disease may result in confluent and multi-lobar consolidation, followed by abscess formation. Silver stains, Steiner, Dieterle, or Warthin-Starry are very useful for demonstration of the bacteria. Modified Brown-Hopps may also be used, although the organisms are weakly Gram-negative, and weakly acid-fast. Immunoperoxidase and immunoflourescent stains are quite sensitive and may be used on paraffin blocks. Electron microscopy may be useful in some cases.

Ehrlichia
Infection by Ehrlichia or ehrlichiosis is transmitted by tick bites; dogs and white-tailed deer are likely reservoirs. Infections are therefore seen during seasons with tick activity. The first human infection was documented in the United States in 1986. Ehrlichia are obligate intracellular bacteria that infect and grow in phagocytic cells, monocyte/macrophage or neutrophils depending on the species. The bacteria are very small, 0.2-1.0um, pleomorphic cocci, that may grow in small clusters within the infected cells as the morulae. The morulae may be demonstrated in the peripheral blood smears by hematologic stains such as Giemsa, Wright, or Leishman, as dark violet-blue cytoplasmic inclusions.

Pathology: The pulmonary findings may be minimal and subtle, and include an interstitial mononuclear infiltrate, with pulmonary hemorrhage, and diffuse alveolar damage developing in severe infections. The alveolar damage may result in a macrophage rich pneumonia. The bacteria can be demonstrated with immunoperoxidase technique in tissue sections and in cytologic preprations, and by PCR technique using blood samples.

Rickettsia
Rickettsial infections are transmitted by bites of infected ticks and mites, or through feces of infected lice and fleas. Rickettsia includes three groups of obligate intracellular bacteria: spotted fever group, typhus group, and scrub typhus group. While the infection is seen world wide, the geographic distribution of a particular rickettsia group is determined by the presence of the arthropod host and its vertebrate host. Morphologically, rickettsiae are very small bacilli, measuring 0.3-1.0um, with cell wall features of a gram-negative organism. Once deposited by the bite of ticks or mites, rickettsia spread via circulation to different organs and attach to the endothelial cell surfaces. Following engulfment by the endothelial cells, the bacteria escape from the phagosome into the cytosol, and start multiplying by binary fission. The pathologic manifestations of rickettsial infection are the result of endothelial injury in various organs.

Pathology: The lungs grossly show edema due to the microvascular injury. Histologically, interstitial pneumonitis with mononuclear cell infiltrates, hyaline membranes and focal alveolar wall necrosis may be seen. Pulmonary microcirculation may show severe compromise, and obvious vasculitis may be seen in some cases. A definitive diagnosis can be made using immunoperoxidase and immunoflourescent stains, and the PCR technique. Demonstration of a >4 fold rise in serum antibody titer is confirmatory. Other test such as immunoflourescent antibody, latex agglutination test, and enzyme immunoassay are also available for confirmation of diagnosis.

Anthrax
Anthrax is a zoonotic disease with worldwide incidence, and primarily a disease of herbivores. The disease was recognized for centuries and reported in early Hebrew and Greek literature. Bacillus anthracis is a large gram-positive, aerobic, non-motile, spore-forming bacillus, found in pairs or short chains in mammalian host. The bacilli measure 1-1.25um in width and 3-5um in length, and have a characteristic "boxcar" or "bamboo rod" appearance. The vegetative forms of anthrax are quickly destroyed by autolytic enzymes, however, the infectious spores can persist in the soil for years. Inhalation of the spores causes a rapidly progressive and usually fatal pulmonary infection. Incubation period is 1-5 days, and severe respiratory distress develops quickly. The chest radiograph often shows a characteristic expansion of the mediastinal space by edema.

Pathology: The hallmark of pathologic lesions in inhalational anthrax is edema and hemorrhage involving the mediastinum and pleural spaces. The lungs show edema and hemorrhagic pneumonia, and gram stain demonstrates large numbers of gram-positive bacilli. The medistinal soft tissues and mediastinal lymph nodes also show edema and hemorrhage. In fatal infections, widespread vasculitis and hemorrhages may be seen in multiple organs. Definitive diagnosis is based on culture and immunohistological staining using a monoclonal antibody against the bacterial cell wall. Electron microscopy with immunogold-conjugated antibody may also be used for diagnosis.

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