Mycobacterium Case File

Mycobacterium Case File

Eugene C.Toy, MD, Cynthia Debord, PHD, Audrey Wanger, PHD, Gilbert Castro, PHD, James D. Kettering, PHD, Donald Briscoe, MD

CASE 14

A 50-year-old man, a recent immigrant from Vietnam, is brought to the emergency room with a cough productive of bloody sputum. He first noticed a cough approximately 2 months ago, but there was not much sputum. In the past several days his sputum production has increased and become mixed with blood. He reports having lost approximately 15 lb in this time frame as well. He also notes that he’s had drenching night sweats 2 or 3 nights a week for the past month. He has a 50-pack-year smoking history but no other medical history. He came to the United States from Vietnam 7 months ago. On examination, he is a thin, frail-appearing male. His vital signs are normal. His head and neck exam is normal. He has no palpable adenopathy in his neck or axilla. His lung exam is notable only for decreased breath sounds diffusely. A chest x-ray shows a cavitary infiltrate of the left upper lobe.

◆ What type of organism is likely to be seen on Gram stain of a sputum sample?

◆ What technique of staining is most commonly used to identify this organism?

◆ What is the histologic characteristic of Langhans cells?

ANSWERS TO CASE 14: Mycobacterium

Summary: A 50-year-old Vietnamese man presents with chronic bloody sputum, weight loss, and a cavitary lesion on chest radiograph, all consistent with tuberculosis.

◆ Likely Gram stain findings of sputum sample: Mycobacterium tuberculosis appear as colorless (“ghost”) cells

◆ Most commonly used staining technique for M. tuberculosis: Acid-fast staining

◆ Histologic characteristics of Langhans cells: Multinucleated giant cells of fused macrophages

CLINICAL CORRELATION

It is estimated that M. tuberculosis infects approximately one-third of the world’s population. Mycobacterium tuberculosis is spread from person to person via aerosolized respiratory droplets that travel to the terminal airways. The bacteria are phagocytized by alveolar macrophages but inhibit destruction by the phago-some and proceed to replicate. Circulating macrophages are attracted to the site of infection and create multinucleated giant cells, composed of fused macrophages called Langhans cells. Extrapulmonary sites are infected through the spread of infected macrophages via lymphatic or hematogenous dissemination. Because of the intracellular nature of M. tuberculosis infections, antibody mediated defenses are relatively ineffective. Persons with conditions of reduced cellular immunity, such as AIDS, alcoholism, or drug abuse, or persons living in crowded, close quarters, such as prisoners, are at increased risk for infection. Organisms can remain dormant in granulomas for many years and reactivate following immunosuppression at a later date. At that time the organisms can infect extrapulmonary sites.

Mycobacterium avium-intracellulare is found in the environment and is an opportunistic pathogen that causes disease in AIDS patients. Disease can range from pneumonia to gastroenteritis to disseminated disease.

Mycobacterium kansasii can clinically mimic pulmonary tuberculosis, but it is most often seen in middle-aged men with prior lung damage such as silicosis or asbestosis. Mycobacterium leprae is acquired by contact with the nine-banded armadillo. Most infections are seen in the southern United States including Texas and Louisiana. Thought to be lepers (skin lesions and deformation of the features of the face), these patients were contained in sanitariums and left to die. Infection can be either of the lepromatous or tuberculous type. Most infections caused by rapidly growing Mycobacteria are chronic wound infections, because this organism is found in the soil.

APPROACH TO SUSPECTED MYCOBACTERIAL INFECTION

Objectives

1. Know the structure and physiology of Mycobacterium.
2. Know the reservoirs, transmission, and diseases caused by Mycobacterium.
3. Know the mechanisms of host defenses and treatments for mycobacterial diseases.

Definitions

Langhans cells: Multinucleated giant cells composed of fused macrophages.

Granuloma: Chronic inflammatory response to either Mycobacterium or fungi, composed of macrophages and multinucleated giant cells.

PPD: Purified protein derivative, prepared from M. tuberculosis antigens inoculated intradermally and a positive reaction is indicative of exposure to M. tuberculosis.

DISCUSSION

Characteristics of Mycobacteria Species

Mycobacterium are small rod-shaped bacilli that stain as ghost cells with Gram stain, but because of the presence of mycolic acids in their cell wall stain with an “acid-fast stain” such as Kinyon or Ziehl-Neelsen. This complex, lipid-rich cell wall also makes the organisms resistant to many commonly used laboratory stains and is responsible for the resistance of this organism to many common detergents and antibiotics.

In general, Mycobacterium species are slow-growing organisms, with a generation time of 15–20 hours, compared to about 1 hour for most bacteria. Mycobacterium can be divided into groups as characterized by Runyon, based on their growth characteristics, particularly pigment production. The photochromogens, which are pigmented only in the presence of light, include M. kansasii, as well as other saprophytic Mycobacterium. The scotochromogens, which are pigmented even without the presence of light, include M. szulgai, as well as the nonpathogenic M. gordonae, which has an orange pigment. The nonchromogens are not pigmented in the light or dark including M. avium-intracellulare, as well as M. haemophilum. The fourth runyon group is composed of the rapidly growing Mycobacteria such as M. fortuitum, M. chelonae, and M. abscessus. The M. tuberculosis complex includes M. tuberculosis, M. africanum, M. ulcerans, and M. bovis, as well as other rarely identified Mycobacterium. These colonies appear buff or tan color and are dry when growing on Lowenstein-Jensen agar. Mycobacterium leprae is not considered in that classification because it cannot be cultured in the laboratory.

One of the virulence factors of M. tuberculosis is cord factor. This can be visualized microscopically as organisms grown in broth culture will demonstrate a ropelike pattern indicating cording. The rapid growing Mycobacterium include M. fortuitum complex, M. chelonae complex, and M. abscessus, as well as other uncommonly isolated nonpathogenic Mycobacterium. These organisms by definition will grow within seven days of subculture onto routine microbiological media such as a blood agar plate.

Diagnosis

Diagnosis of tuberculosis is initially made based on a history (exposure to patient with tuberculosis, immigration, a stay in a jail or homeless shelter) and physical exam in patients with a productive cough, night sweats, and fever. A positive PPD test would indicate exposure to M. tuberculosis and warrant further testing with chest x-ray. Patients with the characteristic upper lobe cavitary lesion would have sputum collected and cultured for Mycobacterium. The classic chest radiograph findings include lower lobe consolidation in active infection, and apical lobe scarring with reactivation.

A fluorescent, direct smear, of the respiratory specimen after decontamination to remove bacterial flora is reported within 24 hours of receipt of the respiratory specimen in the laboratory. Several first early morning deep cough specimens should be collected. Growth of M. tuberculosis on Lowenstein- Jensen (LJ) agar can take 3–8 weeks because of the slow dividing time of the organism; however, with the use of broth medium growth time has been decreased to as short as 1 week. Newly designed automated broth systems read bottles for growth based on carbon dioxide production of the organisms on a daily basis for up to 8 weeks. Bottles which are determined to be positive are stained by Kinyoun stain to visualize the presence of Mycobacterium. Positive bottles can be tested directly for M. tuberculosis, M. kansasii, M. avium-intracellulare, or M. gordonae using DNA probes. Other Mycobacterium species are identified either by routine biochemical tests, which require several weeks, or by high-pressure liquid chromatography, which can speciate Mycobacterium based on mycolic acids extracted from their cell surface. Optimal growth temperature for Mycobacterium species is 35°C (95°F); however, the Mycobacterium that infect the skin such as M. haemophilum grow best at lower temperatures, and organisms such as M. szulgai prefer 42°C (107.6°F). Growth on solid media is also enhanced in the presence of 5–10 percent carbon dioxide.

Treatment and Prevention

Prophylaxis for tuberculosis consists of oral isoniazid for 6–9 months and is given to all patients with a recent conversion of their PPD to positive and a negative chest x-ray. Treatment for tuberculosis based on culture of M. tuberculosis from any patient specimen is initially (first 2 months) with a multiagent regimen based on likely resistance patterns; one such combination is isoniazid, rifampin, ethambutol, and pyrazinamide. Once the results indicate susceptibility to all of the four firstline drugs, treatment can continue with two drugs (usually isoniazid and rifampin) for the remaining 4–6 months. Because of the interaction of rifampin with other drugs, particularly HIV drugs and antifungals, this therapy may need to be individualized.

Prevention of tuberculosis besides prophylactic isoniazid includes isolation of patients in the hospital to prevent spread. Patients with a positive acid-fast smear must remain in isolation until a diagnosis of tuberculosis is ruled out, until they leave the hospital, or following several weeks of appropriate antituberculous therapy with obvious clinical improvement. All known close contacts (family members) of the index case should have a PPD test to determine if they should be given therapy and/or worked up for disease.

Vaccination with BCG, (an attenuated strain of M. bovis) is not routinely performed in the United States because of the comparatively low incidence of tuberculosis. Protection from tuberculosis is not 100 percent with the vaccine and can confuse the results of the PPD for screening of recent converters.

Treatment of the other atypical mycobacteria varies based on the species. Mycobacterium avium-intracellulare is usually treated with clarithromycin or azithromycin and ethambutol plus or minus amikacin. Current treatment for leprosy is dapsone and rifampin for at least 6 months.

COMPREHENSION QUESTIONS

[14.1] An emaciated prisoner in a New York prison began coughing up sputum streaked with blood. Examination of the sputum revealed the presence of acid-fast bacilli. Which of the following would confirm a diagnosis of tuberculosis?

A. Inclusion bodies of the nuclei of macrophages

B. Presence of gram-positive pleomorphic organisms

C. Rough, nonpigmented colonies

D. Rapid growth on Lowenstein-Jensen medium

[14.2] A 45-year-old traveler discovers that he has converted from negative to positive on the tuberculin (PPD) skin test. This indicates which of the following?

A. He has active tuberculosis.

B. He has delayed-type hypersensitivity against M. tuberculosis.

C. He is most likely to be infected with an “atypical” Mycobacterium.

D. He needs to be immediately isolated to prevent spread of M. tuberculosis.

E. He will eventually develop tuberculosis.

[14.3] A 60-year-old man with a chronic cough, bloody sputum, and marked weight loss is diagnosed as having tuberculosis. A “serpentine-like” colonial morphology is noted on Lowenstein-Jensen agar. This latter finding is caused by which of the following factors?

A. A large “slimy” capsule

B. An endotoxin

C. Coagulase

D. Cord factor

E. Wax D

[14.4] A 25-year-old man known to have AIDS experiences a gradual onset of malaise and anorexia, proceeding within a few weeks to photophobia, impaired consciousness, and oculofacial palsy. An acid-fast bacterium with trehalose-6, 6’-dimycolate is isolated. The identity of this organism is which of the following?

A. Mycobacterium fortuitum-chelonei

B. Mycobacterium kansasii

C. Mycobacterium marinum

D. Mycobacterium scrofulaceum

E. Mycobacterium tuberculosis

Answers

[14.1] C. To mount a protective immune response against a specific microorganism requires that the appropriate population of cells play a role in the response. A lipoprotein of M. tuberculosis stimulates a specific “toll-like receptor” on the macrophage. Activated macrophages then synthesize IL-12, which causes differentiation of naïve helper T cells into the Th-1 type of helper T cells that participates in the cell-mediated (delayed hypersensitivity) response. In addition, delayed hypersensitivity (not humoral) reactions are produced against antigens of intracellular pathogens such as M. tuberculosis. Thus, humoral immunity is not protective against M. tuberculosis, and the patient will suffer severe tuberculosis if cell-mediated immunity is not functional. Therefore, an agglutination test for antibodies is useless as M. tuberculosis is an intracellular pathogen and will not elicit a humoral (antibody-formation) immune response that will protect the patient against M. tuberculosis. The growth is usually slow, and the colonies are rough and nonpigmented.

[14.2] B. The purified protein derivative (PPD) skin test, or tuberculin skin test, contains several proteins from M. tuberculosis, which when combined with waxes elicits a delayed hypersensitivity. It does not assess for the status of infection, but only speaks about prior exposure. The clinical presentation and/or chest radiograph would be the next steps in evaluation.

[14.3] D. Virulent strains of M. tuberculosis grow in a characteristic “serpentine” cord-like pattern, whereas avirulent strains do not. Virulence of the organism is correlated with cord factor (trehalose dimycolate).

[14.4] E. Mycobacterium tuberculosis commonly has the trehalose dimycolate factor (see answer to Question 14.3).

MICROBIOLOGY PEARLS ❖ Mycobacterium tuberculosis is a slow growing organism that causes pulmonary infection after close contact with an infected individual. ❖ A positive skin test (PPD) indicates exposure to the organism and not necessarily disease. ❖ Mycobacterium species stain positive with an acid-fast stain because of components in their cell wall. ❖ Initial therapy for tuberculosis requires multiple agents to avoid the development of resistance, and culture susceptibilities will dictate which agents should be continued.

REFERENCES

Haas DW. Mycobacterium tuberculosis. In: Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases, 5th ed. Philadelphia, PA: Churchill Livingstone, 2000:2576–607. 

Murray PR, Rosenthal KS, Pfaller MA. Mycobacterium. In: Rosenthal KS, Kobayashi GS, Pfaller MA. Medical Microbiology, 5th ed. St. Louis, MO: Mosby, 2005:297–310.

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