Mycobacteriology Laboratory Practices

Mycobacterium tuberculosis and Nontuberculous Mycobacteria

• Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM) are a group of bacteria with differing clinical significances
• Mycobacterium tuberculosis causes tuberculosis (TB)
• Mycobacterium leprae causes leprosy (Hansen's disease)

Objectives

• Compare general characteristics of mycobacteria to other bacteria
• Describe the clinical significance of nontuberculous mycobacteria
• Discuss safety precautions in mycobacteriology labs
• Describe appropriate specimen collection and processing for mycobacteria recovery
• Justify specimen digestion and decontamination for mycobacterial isolation
• Describe the principles and procedures for stains used to demonstrate mycobacteria
• Compare different culture media used for mycobacterial isolation
• Discuss different tests used to identify mycobacteria
• Compare continuous monitoring systems with conventional media for detecting mycobacterial species
• Develop isolation and identification protocol for M. tuberculosis from a sputum specimen
• Discuss clinical disease caused by M. tuberculosis
• Discuss the use of the tuberculin test

Genus Mycobacteria

• Species cause dreaded diseases like TB and leprosy
• Immunocompromised patients may experience resurgence of TB or diseases caused by other mycobacteria
• Some environmental saprophytes (atypical mycobacteria or MOTT) can cause TB-like human infections

General Characteristics

• Slender, slightly curved or straight rods
• High lipid content (mycolic acid) in cell walls, which makes them resistant to staining (acid-fast bacilli, AFB)
• Strictly aerobic
• Pathogenic ones grow slowly, requiring 2-6 weeks on complex media
• M. leprae does not grow in vitro

Mycobacterium tuberculosis (MTB)

• Robert Koch first described M. tuberculosis in 1882
• WHO estimated 12 million people worldwide had TB in 2011
• Usually affects the respiratory tract

Primary Tuberculosis

• Development depends on patient's cellular immune response, exposure amount, and strain virulence

• Acquired from active cases through airborne droplets (1-5 µm) during coughing/talking

• MTB are phagocytized by alveolar macrophages

• Capable of intracellular multiplication

• In those with adequate CMI, T cells arrive within 4-6 weeks, destroying intracellular mycobacteria, leading to lesion regression and healing

• Granuloma formation may occur in some, leading to healing

• Without granuloma, lesions heal without significant pathology

• After primary infection, bacilli may remain viable in granulomas for months/years and potentially reactivate

• Clinical diagnosis of primary TB is typically limited to signs/symptoms and a positive PPD skin test

• Some infected individuals develop progressive pulmonary disease due to failed CMI, leading to bacilli multiplication

• 10% of young adults may progress to active disease from primary infection

Reactivation Tuberculosis

• Risk of reactivation TB is 3.3% during the first year of a positive PPD
• Disease development is slow (insidious) and characterized by fever, shortness of breath, night sweats, chills, fatigue, anorexia, and weight loss
• 20% of individuals are asymptomatic
• Diagnosis confirmed by stained smear, sputum/gastric aspirate or bronchoscopy specimens (95% accuracy)

Extrapulmonary Tuberculosis

• Less common than pulmonary TB
• Miliary Tuberculosis involves seeding many organs outside the pulmonary tree, dispersed through hematogenous spread
• Children account for many cases of miliary tuberculosis
• Resolution is common, but AFB may be found in pleural fluid (20-50% of cases) and higher yield with pleural biopsies

Identification of Mycobacterium tuberculosis

• Colonies grow slowly, are typically raised with a dry, rough appearance, and are non-pigmented, buff colored
• Cord formation due to cord factor elaboration
• Optimal growth at 35-37°C

Biochemical reactions

• Niacin test positive (M. tuberculosis)
• Catalase production differentiates strains (isoniazid-resistant strains are catalase negative.)
• Inhibited by Nitroimidazopyran or p-nitroacetylamine-B-propiophenone (NAP) . Distinguished from M. bovis.

Treatment

• Involves multiple anti-mycobacterial agents

Multidrug-Resistant Mycobacterium tuberculosis (MDR-TB)

• Drug resistance is usually acquired by spontaneous mutation
• MDR-TB is resistant to at least INH and rifampin
• XDR-TB is resistant to INH, rifampin, and at least one injectable second-line anti-TB drug plus one fluoroquinolone class drug

Mycobacterium bovis

• Primarily infects cattle and other ruminants (also dogs, cats, pigs, parrots and humans)
• Human disease resembles TB
• Belongs to the MTB complex
• Grows very slowly on egg-based media, similar to M. tuberculosis, though slower to mature
• Niacin-negative

Clinical Significance of Nontuberculous Mycobacteria (NTM)

• Mostly found in soil and water
• Opportunistic pathogens
• Cause chronic pulmonary disease mimicking TB
• Some types associated with skin infections
• Infections not considered person-to-person transmissible

Slowly Growing Mycobacteria

• Photochromogens (e.g., M. kansasii, M. marinum)
• Scotochromogens (M. scrofulaceum)
• Non-photochromogens/nonchromogens (M. avium-intracellulare)

Mycobacterium avium complex (MAC)

• Epidemiology: common in environmental saprophytes (soil, water, etc).
• Clinical Infections: pulmonary disease like MTB, common with AIDS
• Laboratory Diagnosis: colonies grow slowly, microscopic appearance—short coccobacilli, uniformly stained, no beading or banding, heat stable catalase +, nucleic acid probes available

Mycobacterium kansasii

• Epidemiology: second to MAC in prevalence as cause of NTM lung disease. Isolated from water, but contagious.
• Clinical Infections: Upper lung lobes, with cavitation and scarring
• Laboratory diagnosis: Slow-growth organisms, long rods with cross-banding, some cording, and strongly catalase-positive

Mycobacterium marinum

• Implicated in fish diseases and human cutaneous infections (e.g. tender red subcutaneous nodules, swimming pool granulomas
• Photochromogen
• Microscopic appearance: long rods with cross barring
• Niacin and urease-positive

Mycobacterium ulcerans

• Rare cause of mycobacteriosis (Buruli ulcer)
• Worldwide prevalence, third most common behind TB and leprosy
• Painless skin nodule after previous trauma, no significant systemic symptoms, and non-photochromogenic colonies appear with weeks

Mycobacterium scrofulaceum

• Most common cause of cervical lymphadenitis in children before MAC.
• Infection causes swollen lymph nodes in the neck near the jaw
• Colonies are scotochromogenic; urease, catalase are positive; do not reduce nitrate
• Microscopic: uniformly stained AFB, medium-to-long rods

Laboratory Diagnosis

• Techniques: AFB detection, cultural methods (rate of growth, colony morphology, pigmentation, nutritional requirements, optimal incubation temperature, biochemical test results; other rapid techniques like broth-based culture systems, species-specific probes, PCR (increase the sensitivity of nucleic acid probes), HPLC (distinguishing mycobacterial species).

• Safety Precautions: proper handling of specimens; specialized lab settings (ventilation systems, negative air pressure and safety cabinets); use of personal protective equipment

• Specimen collection and processing are crucial; specific methods for different specimen types (sputum, gastric aspirate, urine, stool, blood, tissue).

Digestion and Decontamination of Specimens

• Specimens (sputum, gastric, BAL, bronchial washing, transtracheal aspirates) often require digestion and decontamination to concentrate mycobacteria
• Decontaminate to reduce contaminants by using chemicals (NaOH, N-Acetyl-L-cysteine). May use selective media to reduce need for harsh decontamination processes

Biochemical test

• Niacin test
• Nitrate reduction
• Catalase

Molecular Methods

• Nucleic acid amplification tests (NAATs) using PCR (speed up diagnosis, detect TB and resistance to anti-TB drugs)

• GeneXpert MTB/RIF (faster results within 2 hours—detects MTB.)

Immunodiagnosis of MTB Infection

• Tuberculin Skin Test: PPD (purified protein derivative) injected intradermally. Reacts based on prior exposure and cell-mediated immunity, not always specific to M. tuberculosis
• Quantiferon-TB Gold Assay and the T.SPOT.TB: measures CMI (cell-mediated immunity) response to mycobacterial antigens. More sensitive and specific than tuberculin skin tests for children.

Specimens Storage

• Methods and times needed to store different samples for further testing

Treatment Susceptibility

• Methods need to test if patient is resistant or susceptible to the primary agents or further types of molecular tests
• Need to run tests for resistant strains quickly for optimal treatment

Thank You