Chapter 22: Legionella, Bartonella, and Unusual Bacterial Pathogens

A widely publicized outbreak of pneumonia in persons attending an American Legion convention in Philadelphia in 1976 prompted investigations that defined Legionella pneumophila and the legionellae. Other outbreaks of respiratory illness caused by related organisms since 1947 have been diagnosed retrospectively. Several dozen species of Legionella exist, some with multiple serogroups. L pneumophila is the major cause of disease in humans; Legionella micdadei and a few other species sometimes cause pneumonia. The other legionellae are rarely isolated from patients or have been isolated only from the environment.

Morphology and Identification

L pneumophila is the prototype bacterium of the group. Legionellae of primary medical importance are listed in Table 22-1.

A. Typical Organisms

Legionellae are fastidious, aerobic gram-negative bacteria that are 0.5–1 μm wide and 2–50 μm long (Figure 22-1). They often stain poorly by Gram’s method and are not easily seen in stains of clinical specimens. Gram-stained smears should be made for suspect Legionella growth on agar media. Basic fuchsin (0.1%) should be used as the counterstain because safranin stains the bacteria very poorly. Silver stains, such as Warthin-Starry and Dieterle, can be used to detect legionellae in embedded tissues. Of note, L micdadei may be acid-fast stain positive.

B. Culture and Growth Characteristics

Legionellae can be grown on complex media such as buffered charcoal yeast extract agar with α-ketoglutarate, L-cysteine, and iron (BCYE) at a pH of 6.9, temperature of 35°C, and 90% humidity. Antibiotics can be added to make the medium selective for Legionella species. The charcoal acts as a detoxifying agent. Legionellae grow slowly; visible colonies are usually present after 3 days of incubation. Colonies that appear after overnight incubation are not Legionella species. Colonies are round or flat with entire edges. They vary in color from colorless to iridescent pink or blue and are translucent or speckled. Variation in colony morphology is common, and the colonies may rapidly lose their color and speckles. Many other genera of bacteria grow on BCYE medium and must be differentiated from Legionella by Gram staining and other tests.

Suspicious colonies require definitive identification by methods other than biochemical assessment since legionellae are biochemically inert. Confirmatory tests include direct fluorescent antibody tests, 16SrRNA gene sequencing, and the use of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS).

The legionellae are catalase positive. L pneumophila is oxidase positive; the other legionellae are variable in oxidase activity. L pneumophila hydrolyzes hippurate; the other legionellae do not. Most legionellae produce gelatinase and β-lactamase; L micdadei produces neither gelatinase nor β-lactamase.

Antigens and Cell Products

Antigenic specificity of L pneumophila is thought to be attributable to complex antigenic structures. There are at least 16 serogroups of L pneumophila; serogroup 1 was the cause of the 1976 outbreak of Legionnaires’ disease and remains the most common serogroup isolated from humans. Legionella species cannot be identified by serogrouping alone because there is cross-reactive antigenicity among different species. Occasionally, other gram-negative bacteria may also cross-react with L pneumophila antisera.

The legionellae produce distinctive 14- to 17-carbon branched-chain fatty acids. Gas-liquid chromatography can be used to help characterize and determine the species of legionellae.

The legionellae make proteases, phosphatase, lipase, DNase, and RNase. A major secretory protein, a metalloprotease, has hemolytic and cytotoxic activity; however, this protein has not been shown to be a required virulence factor.

Pathology and Pathogenesis

Legionellae are ubiquitous in warm, moist environments. They are found in lakes, streams, and other bodies of water. They can multiply in free-living amebas and can coexist with them in biofilms (see Epidemiology and Control). Infection of debilitated or immunocompromised humans commonly follows inhalation of the bacteria from aerosols generated from contaminated air-conditioning systems, shower heads, and similar water sources. L pneumophila usually produces a lobar, segmental, or patchy pulmonary infiltration. Histologically, the appearance is similar to that produced by many other bacterial pathogens. Acute pneumonia involving the alveoli is present with a dense intra-alveolar exudate of macrophages, polymorphonuclear leukocytes, red blood cells, and proteinaceous material. Most of the legionellae in the lesions are within phagocytic cells. There is little interstitial infiltration and little or no inflammation of the bronchioles and upper airways.

Knowledge of the pathogenesis of L pneumophila infection comes from study of isolated cells from humans and from study of susceptible animals such as guinea pigs.

L pneumophila readily enters and grows within human alveolar macrophages and monocytes and is not effectively killed by polymorphonuclear leukocytes. Legionella species do not require opsonization by C3b or antibody to enter macrophages. A virulence factor important for macrophage invasion is the Mip protein, which promotes adherence and phagocytosis. Inside the cell, the individual bacteria are contained within phagosomal vacuoles (Legionella-containing vacuole, LCV), but the defense mechanisms of the macrophages stop at that point. Instead, the LCV fails to fuse with lysosomal granules. The phagocyte oxidative metabolic burst is reduced. The LCV does not acidify as much as phagosomes containing other ingested particles. Ribosomes, mitochondria, and small vesicles accumulate around LCVs, preventing recognition by the cellular immune system. In addition to this process, phagosome survival and organism replication are facilitated by elaboration of a type IV secretion system call Dot/Icm which is essential for L pneumophila virulence. The bacteria multiply within the vacuoles until they are numerous, the cells are destroyed, the bacteria are released, and infection of other macrophages then occurs. The presence of iron (transferrin iron) is also essential for the process of intracellular growth of the bacteria.

Clinical Findings

Asymptomatic infection is common in all age groups, as shown by elevated titers of specific antibodies. The incidence of clinically significant disease is highest in men after age 55 years. Disease in children has been reported but remains rare. Factors associated with high risk include smoking, alcohol misuse, diabetes mellitus, chronic bronchitis and emphysema or cardiovascular disease, steroid and other immunosuppressive treatment (as in renal transplantation), cancer chemotherapy, and, more recently, as a complication of anti–tumor necrosis factor (TNF)-α therapy, especially infliximab or adalimumab. When pneumonia occurs in patients with these risk factors, Legionella should be investigated as the cause.

Infection may result in nondescript febrile illness of short duration or in a severe, rapidly progressive illness with high fever, chills, malaise, nonproductive cough, hypoxia, diarrhea, and delirium. Chest radiography reveals patchy, often multilobar consolidation. Immunocompromised patients may develop cavitary pneumonia and pleural effusions. There may be leukocytosis, hyponatremia, hematuria (and even renal failure), or abnormal liver function. During some outbreaks, the mortality rate has reached 10%. The diagnosis is based on the clinical picture and exclusion of other causes of pneumonia by laboratory tests. Demonstration of Legionella species in clinical specimens can rapidly yield a specific diagnosis. The Legionella urinary antigen test may be used early in the course of infection with L pneumophila serogroup 1 and is very helpful when the result is positive. The diagnosis can also be made by culture for Legionella species or by serologic tests, but results of these tests are often delayed beyond the time when specific therapy must be started.

L pneumophila also produces a disease called “Pontiac fever” after the clinical syndrome that occurred in an outbreak in Michigan. The syndrome is characterized by fever and chills, myalgia, malaise, and headache that develop over 6–12 hours and persist for 2–5 days. Dizziness, photophobia, neck stiffness, and confusion also occur. Respiratory symptoms are much less prominent in patients with Pontiac fever than in those with Legionnaires’ disease and include mild cough and sore throat. This illness is self-limited and does not require treatment with antibiotics.

Diagnostic Laboratory Tests

A. Specimens

In human infections, the organisms can be recovered from expectorated sputum (when available), bronchial washings, pleural fluid, lung biopsy specimens, or (rarely) blood. Isolation of Legionella species from sputum is more difficult because of the predominance of bacteria of the normal microbiota and because often the cough is dry. Legionella species are rarely recovered from other anatomic sites.

B. Smears

Legionellae are not demonstrable in Gram-stained smears of clinical specimens. Direct fluorescent antibody tests of specimens can be diagnostic, but the test has low sensitivity compared with culture and is rarely done directly on clinical specimens. Silver stains are sometimes used on tissue specimens.

C. Culture

Specimens are cultured on BCYE agar with and without antibiotics (see earlier discussion). Cultured organisms can be rapidly identified by immunofluorescence staining. MALDI-TOF MS has the potential to provide rapid diagnosis of culture isolates.

D. Specific Tests

Sometimes Legionella antigens can be demonstrated in the patient’s urine by immunologic methods. The urine antigen test is specific for L pneumophila serogroup 1. Thus, the Legionella urine antigen test is not useful to diagnose 20–70% of Legionella species infections, depending on geographic location, and should not be relied on as the sole test to rule out Legionella infections. Molecular assays, such as polymerase chain reaction (PCR), that amplify genes such as mip and 16SrRNA, among others, have been used by laboratories capable of developing and verifying their own assays. However, specificity has been an issue related to contamination of reagents with Legionella DNA found in water sources. In the United States, there are no FDA-cleared assays that are available for Legionella detection.

E. Serologic Tests

Levels of antibodies to legionellae rise slowly during the illness. Serologic tests have a sensitivity of 60–80% and a specificity of 95–99%. Serologic tests are most useful in obtaining a retrospective diagnosis in outbreaks of Legionella infections.

Immunity

Infected patients make antibodies against Legionella species, but the peak antibody response may not occur until 4–8 weeks after infection. The roles of antibodies and cell-mediated responses in protective immunity in humans have not been defined. Animals challenged with sublethal doses of virulent L pneumophila, avirulent L pneumophila, or a major secretory protein vaccine are immune to subsequent lethal doses of L pneumophila. Both humoral and cell-mediated immune responses occur. The cell-mediated response is important in protective immunity because of the intracellular infection and growth of Legionella.

Treatment

L pneumophila are intracellular parasites of macrophages, other phagocytic cells, and probably of other human cells as well. Other Legionella species may also show significant growth within human macrophages. Thus, antimicrobials useful for treatment of Legionella infections must enter the phagocytes and have biological activity there. Macrolides (erythromycin, azithromycin, telithromycin, and clarithromycin), quinolones (ciprofloxacin and levofloxacin), and tetracyclines (doxycycline) are effective. β-Lactams, monobactams, and aminoglycosides are not effective; in addition, many legionellae make β-lactamases. Prolonged therapy, up to 3 weeks, may be required depending on the clinical situation. Therapy should not be discontinued until the patient has been afebrile for 48–72 hours.

Epidemiology and Control

Peak season for Legionella infections is late summer to autumn. Travel, particularly on cruise ships, may be a risk factor. Transmission is usually the result of inhalation or ingestion followed by aspiration of aerosols from contaminated water systems. The natural habitats for legionellae are lakes, streams, rivers, and especially thermally heated bodies of water and soil. Legionellae grow best in warm water in the presence of amebas and water bacteria. They proliferate in amebas much as they do in pulmonary macrophages in the lung. When harsh environmental conditions occur and the amebas encyst, the amebas and legionellae both survive until better growth conditions occur, allowing excystment. The legionellae, amebas, and other microorganisms exist in biofilms; the legionellae go into a sessile state. The legionellae survive water treatment processes, and small numbers enter the water distribution systems, where they proliferate.

Cooling towers and evaporative condensers can be heavily contaminated with L pneumophila. Presumably, aerosols exiting such towers or condensers spread the organisms to susceptible persons. Similarly, there are links between contamination of residential water systems and community-acquired Legionnaires’ disease and between contamination of hospital water systems and nosocomial L pneumophila infection. Hyperchlorination and superheating of water can help control the multiplication of legionellae in water and in air-conditioning systems. More effective measures include point of use filters, copper–silver ionization, and possibly chlorine dioxide or monochloramine (see Lin et al, 2011).

Concept Checks

• Legionella species are ubiquitous, poorly staining gram-negative bacteria that inhabit fresh water and a variety of potable water systems, where they survive within amebae and biofilms. Humans acquire infections from inhalation of contaminated water.

• There are more than 50 species of Legionella, but most infections are caused by L pneumophila serogroup 1. Patients at risk for infection include those who are immunocompromised, such as bone marrow and solid organ transplant recipients; patients who smoke or have chronic lung disease; and patients with diabetes mellitus.

• Legionnaire’s disease is a multisystem infection that includes pneumonia, gastrointestinal symptoms, delirium, and a variety of laboratory abnormalities. Legionella pneumonia is indistinguishable from other bacterial causes of lower respiratory tract infections.

• Diagnosis rests on a strong clinical specimen and the use of Legionella urinary antigen testing and culture. Serology is largely retrospective and insensitive. Molecular diagnostic tests are not widely available and may lack specificity.

• Legionella species are intracellular pathogens, and hence only antibiotics capable of intracellular penetration such as macrolides and fluoroquinolones should be used for treatment.

• Hospitals that care for immunocompromised patients should monitor potable water systems for the presence of Legionella species and treat the water if they are found. Local public health authorities and the Centers for Disease Control and Prevention can provide guidance for testing and treatment.

The three most prevalent species of medical importance in the genus Bartonella are Bartonella bacilliformis, the cause of Oroya fever and verruga peruana; Bartonella quintana, the cause of trench fever and some cases of bacillary angiomatosis; and Bartonella henselae, which causes cat-scratch disease and has also been associated with bacillary angiomatosis. These diseases have many common characteristics. There is an additional small set of Bartonella species and subspecies that have rarely been associated with human disease, primarily endocarditis, and these are Bartonella elizabethae, Bartonella vinsonii subsp. berkhoffi, Bartonella vinsonii subsp. arupensis, Bartonella koehlerae, and Bartonella alsatica. There is a larger set associated with animals, and transmission to humans from these animal reservoirs has not yet been described.

The Bartonella species are intracellular, gram-negative rods that are pleomorphic, slow growing, and difficult to isolate in the laboratory. They can be seen in infected tissues stained with the Warthin-Starry silver impregnation stain.

Bartonella Bacilliformis

There are two stages of B bacilliformis infection. The initial stage is Oroya fever, a serious infectious anemia. The second is the eruptive stage, verruga peruana, which commonly begins 2–8 weeks later, although verrugae may also occur in the absence of Oroya fever.

Oroya fever is characterized by the rapid development of severe anemia caused by red blood cell destruction, enlargement of the spleen and liver, and hemorrhage into the lymph nodes. Masses of bartonellae fill the cytoplasm of cells lining the blood vessels, and endothelial swelling may lead to vascular occlusion and thrombosis. The mortality rate of untreated Oroya fever can be as high as 85%. The diagnosis is made by examining stained blood smears and blood cultures in semisolid medium.

Weeks to months following acute infection, a second stage of infection called verruga peruana, characterized by vascular nodular skin lesions that occur in successive crops, appears. This infection lasts for about 1 year and produces little systemic reaction and no fatalities. Mucosal and internal lesions have been described. Bartonellae can be seen in the granulomas; blood culture results are often positive, but there is no anemia.

B bacilliformis produces an extracellular protein called deformin that promotes deformity (indentation) of red blood cell membranes, and flagella provide the organisms with the mechanical force to invade red blood cells. Replication of the organism occurs within an endocytic vacuole facilitated by outer membrane proteins and erythrocyte membrane fragments created at the time of attachment and membrane deformity. B bacilliformis also invades endothelial cells and other types of human cells in vitro.

Bartonellosis is limited to the mountainous areas of the American Andes in tropical Peru, Colombia, and Ecuador, and is transmitted by sandflies of the genus Lutzomyia.

B bacilliformis grows in semisolid nutrient agar containing 10% rabbit serum and 0.5% hemoglobin. After 10 days or more of incubation at 28°C, turbidity develops in the medium, and rod-shaped and granular organisms can be seen in Giemsa-stained smears.

Ciprofloxacin, doxycycline, macrolides, or sulfamethoxazole–trimethoprim given for at least 10 days has been used to successfully treat patients. Parenteral therapy can be used if the patient is unable to absorb oral medication. Chloramphenicol for 14 days has been used to effectively treat B bacilliformis infections particularly in South America. Coupled with blood transfusions, when indicated, antimicrobial therapy greatly reduces the mortality rate. Control of the disease depends on elimination of the sandfly vectors; insecticides, insect repellents, and elimination of sandfly breeding areas are of value. Prevention with antibiotics may be useful.

Bartonella henselae and Bartonella quintana

A. Cat-Scratch Disease

Cat-scratch disease is usually a benign, self-limited illness manifested by fever and lymphadenopathy that develop 1–3 weeks after contact with a cat (usually a scratch, lick, bite, or perhaps a flea bite). A primary skin lesion (papule or pustule) develops at the site. The patient usually appears well but may have low-grade fever and occasionally headache, malaise, and sore throat. The regional lymph nodes (axillary, epitrochlear, or cervical most commonly) are markedly enlarged and sometimes tender, and they may not subside for several weeks or even months. They may suppurate and discharge pus. Atypical cases (5–10%) may be characterized by preauricular lymphadenopathy and conjunctivitis (Parinaud’s oculoglandular syndrome). More serious systemic features such as meningitis, encephalopathy, bone lesions, and retinitis have been described. More than 22,000 cases a year are thought to occur in the United States.

The diagnosis of cat-scratch disease is based on (1) a suggestive history and physical findings; (2) aspiration of pus from lymph nodes that contain no bacteria culturable by routine methods; and (3) characteristic histopathologic findings with granulomatous lesions, which may include bacteria seen on silver-impregnated stains. A positive skin test result has also been included as a criterion, but is of historical interest only. A titer of 1:64 or greater in a single serum in the indirect fluorescent antibody (IFA) test strongly supports the diagnosis, but development of a diagnostic titer may be delayed or may not occur in immunocompromised patients. Enzyme immunoassays are available but may be less sensitive than IFA.

Cat-scratch disease is caused by B henselae, a small, pleomorphic, gram-negative rod present mainly in the walls of capillaries near follicular hyperplasia or within microabscesses. The organisms are seen best in tissue sections stained with Warthin-Starry silver impregnation stain; they may also be detected by immunofluorescent stains. Culture of B henselae is generally not recommended for this relatively benign disease.

The reservoir for B henselae is domestic cats, and one-third of cats or more (and possibly their fleas) may be infected. Contact with infected cats through skin lesions is thought to transmit the infection.

Cat-scratch disease occurs commonly in immunocompetent people and is usually self-limited. Treatment is mainly supportive: reassurance; hot, moist soaks; and analgesics. Aspiration of pus or surgical removal of an excessively large lymph node may ameliorate symptoms. While anecdotal reports demonstrate that tetracycline, azithromycin, trimethoprim–sulfamethoxazole, rifampin, gentamicin, or fluoroquinolone therapy may be helpful, more recent analyses do not support treatment with antibiotics.

B. Bacillary Angiomatosis

Bacillary angiomatosis is a disease predominantly of immunosuppressed individuals, particularly individuals with AIDS. Rare cases occur in immunocompetent persons. Bacillary angiomatosis is characterized histopathologically as circumscribed lesions with lobular capillary proliferation and round, open vessels with cuboidal endothelial cells protruding into the vascular lumen. A prominent finding is epithelioid histiocytes surrounded by a loose fibromyxoid matrix. The pleomorphic bacilli can be seen in the subendothelial tissue when stained with the Warthin-Starry silver impregnation stain. The lesions may be infiltrated by polymorphonuclear leukocytes.

In its common form, bacillary angiomatosis presents as an enlarging red (cranberry-like) papule, often with surrounding scale and erythema. The lesions enlarge and may become several centimeters in diameter and ulcerate. There may be single or many lesions. The clinical appearance is often similar to that of Kaposi sarcoma in AIDS patients, but the two diseases are different histologically. Bacillary angiomatosis occurs in virtually every organ. Involvement of the liver (and spleen) is characterized by a proliferation of cystic blood-filled spaces surrounded by a fibromyxoid matrix containing the bacteria; this form of the disease is called peliosis hepatis and is usually accompanied by fever, weight loss, and abdominal pain. A bacteremic form of infection with the nonspecific signs of malaise, fever, and weight loss also occurs.

The diagnosis is confirmed by the characteristic histopathologic findings and demonstration of the pleomorphic bacilli on silver-stained sections. B henselae and B quintana can be isolated by direct culture of biopsies of involved tissue carefully obtained so that no contaminating skin bacteria are present. The biopsy specimens are homogenized in supplemented tissue culture medium and inoculated onto fresh chocolate agar and heart infusion agar with 5% rabbit blood. Cultures of blood obtained by the lysis centrifugation method can be inoculated onto the same media. The cultures should be incubated in 5% CO₂ at 36°C for a minimum of 3 weeks. Specimens can also be cultured on eukaryotic tissue culture monolayers. Biochemically, B henselae and B quintana are relatively inert, including negative catalase and oxidase reactions and negative carbohydrate utilization tests. Enzyme activity can be seen with amino acid substrates by methods to test for preformed enzymes. Definitive identification is obtained by sequencing all or part of the 16S ribosomal RNA gene amplified by the PCR. Because of the difficulty in recovering Bartonella species from clinical material and the insensitivity to date of molecular methods, serologic testing is still considered by many to be the best option. IFA tests are the most frequently used.

Bacillary angiomatosis is treated with oral erythromycin (drug of first choice) or doxycycline (plus gentamicin for very ill patients) for a minimum of 2 months. The often rapid response of skin lesions to erythromycin is believed to be due to its anti-inflammatory and anti-angiogenic effects. Relapses are common but can be treated by the same initial drug regimen.

C. Trench Fever

Trench fever (also known as quintan fever) is characterized by sudden onset of fever accompanied by headache, malaise, restlessness, and shin pain. Symptoms coincide with release of B quintana in blood every 3–5 days with each episode lasting 5 days. A prominent affliction seen during World War I, B quintana is now more frequently seen as a cause of culture-negative endocarditis and bacteremia in homeless individuals.

The reservoir for B henselae usually is the domestic cat, and patients with this organism as the etiology of bacillary angiomatosis often have contact with cats or histories of cat flea bites. The only known reservoirs for B quintana are humans and the body louse.

Concept Checks

• Bartonella species are small gram-negative rods that are found among animals, humans, and their vectors.

• The human pathogens include B bacilliformis, which causes acute Oroya fever and chronic verruga peruana primarily among populations in the Andes, and B quintana, which is responsible for trench fever, endocarditis, and bacillary angiomatosis. B henselae can cause endocarditis, cat-scratch disease, and bacillary angiomatosis. Infection is acquired by the bite or scratch from a cat or perhaps a bite from cat fleas.

• Diagnosis of B henselae infections may be difficult because they are slow growing and grow best on media containing rabbit blood or chocolate agar. Demonstration of organisms in tissue using Warthin-Starry stains is possible. Serologic diagnosis is the mainstay.

• Treatment of Bartonella infections includes azithromycin or other macrolides, fluoroquinolones, and doxycycline.

Streptobacillus moniliformis is an aerobic, gram-negative, highly pleomorphic organism that forms irregular chains of bacilli interspersed with fusiform enlargements and large round bodies. It grows best at 37°C in media containing serum protein, egg yolk, or starch, but ceases to grow at 22°C. L forms can easily be demonstrated in most cultures of the organism. Subculture of pure colonies of L forms in liquid media often yields the streptobacilli again. Previously believed to be the only species in the genus, recently it has been joined by a novel species officially named Streptobacillus hongkongensis sp. nov. S moniliformis is a normal inhabitant of the throats of rats, and humans can be infected by rat bites. The human disease (rat-bite fever) is characterized by septic fever, blotchy and petechial rashes, and very painful polyarthritis. Other types of presentations include bacteremia, endocarditis, and abscesses. Diagnosis rests on cultures of blood, joint fluid, or pus; on mouse inoculation (not done in clinical laboratories); and on serum agglutination tests. This organism can also produce infection after being ingested in milk. The disease is called Haverhill fever and has occurred in epidemics.

The reservoir for S hongkongensis is unknown. This organism has been recovered from a peritonsillar abscess and a septic elbow from separate patients in Hong Kong.

Penicillin, third-generation cephalosporins, and some fluoroquinolones all have activity against S moniliformis and S hongkongensis.

Rat-bite fever of somewhat different clinical appearance (sodoku) is caused by Spirillum minor (see Chapter 24).

Whipple disease is characterized by fever, abdominal pain, diarrhea, weight loss, and migratory polyarthralgia most commonly in middle-aged men. The primary site of involvement is the small intestine and mesenteric lymph nodes, but any organ can be affected; most notably, musculoskeletal, neurologic, cardiac, and ophthalmic manifestations are described. Histologically, there is a prominent macrophage infiltration and glycoprotein deposition. Characteristic vacuoles within the macrophage that stain with periodic acid-Schiff (PAS) stain (foamy macrophages) are pathognomonic of the disease. The intracellular and extracellular PAS-positive materials are bacilli. Historically, routine cultures of clinical specimens have been negative, but more recently the organism has been cultured in association with eukaryotic cells (human fibroblasts, deactivated peripheral blood monocytes). Before the organism was successfully cultured, PCR amplification of bacterial 16S ribosomal RNA allowed identification of a unique sequence from the bacteria in the lesions. Phylogenetic analysis has shown the organism is a gram-positive actinomycete not closely related to any known genus. The organism has been named Tropheryma whipplei. The diagnosis of Whipple disease is by PCR amplification of an appropriate specimen (bowel biopsy, brain biopsy, etc) for T whipplei.