Mycobacteriology Laboratory Practices
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Questions and Answers

What is an essential step to prevent dehydration of tissue samples not processed immediately?

  • Add 15-20 ml of sterile saline
  • Add 10-15 ml of sterile saline (correct)
  • Add 5-10 ml of sterile saline
  • Add 20 ml of sterile saline

    • Histopathologic changes like caseating granuloma are specific for mycobacterial disease.

    False

    What should be done with potentially infected clinical samples within 24 hours?

    Transfer to laboratory

    A ___________ culture system is a more rapid technique for identifying mycobacteria.

    <p>broth-based</p> Signup and view all the answers

    Match the features used to differentiate species within the genus Mycobacteria with their descriptions:

    <p>Rate of growth = Speed of colony development Colony morphology = Physical appearance of colonies Pigmentation = Color changes in colonies Nutritional requirements = Needed nutrients for growth Optimal incubation temperature = Ideal temperature for growth Biochemical test results = Chemical reactions observable in cultures</p> Signup and view all the answers

    What method can increase the sensitivity of nucleic acid probes for identifying mycobacteria?

    <p>Polymerase chain reaction (PCR)</p> Signup and view all the answers

    It is not necessary to have special safety equipment in a mycobacteriology laboratory.

    <p>False</p> Signup and view all the answers

    The mycobacteriology laboratory should have a __________ ventilation system.

    <p>noncirculating</p> Signup and view all the answers

    Which of the following is the single most important piece of equipment in a mycobacteriology laboratory?

    <p>Biological Safety Cabinet</p> Signup and view all the answers

    All potentially infectious materials should be left uncovered when outside the safety cabinet.

    <p>False</p> Signup and view all the answers

    What is the recommended frequency for testing and recertifying safety cabinets?

    <p>At least yearly</p> Signup and view all the answers

    Laboratories should maintain _____ room air changes per hour to effectively remove airborne particles.

    <p>6-12</p> Signup and view all the answers

    Match the disinfectant to its appropriate concentration:

    <p>Sodium hypochlorite = 0.1%-0.5% 5% phenol = 10-30 minutes contact time 3-8% formaldehyde = 30 minutes contact time 2% alkaline glutaraldehyde = At least 30 minutes contact time</p> Signup and view all the answers

    What should be used to cover the work surface to reduce the accidental creation of infectious aerosols?

    <p>Towel or absorbent pad soaked in disinfectant</p> Signup and view all the answers

    UV light can be turned on when the safety cabinet is in use.

    <p>False</p> Signup and view all the answers

    What is the purpose of using aerosol-free safety carriers when centrifuging specimens?

    <p>To prevent the dispersal of infectious aerosols</p> Signup and view all the answers

    What is the major portal of entry for Mycobacterium leprae?

    <p>Respiratory tract</p> Signup and view all the answers

    Tuberculoid leprosy is characterized by a severe and extensive form of the disease.

    <p>False</p> Signup and view all the answers

    What are the two major forms of leprosy?

    <p>Tuberculoid leprosy and Lepromatous leprosy</p> Signup and view all the answers

    The earliest symptoms of leprosy include a slightly hypopigmented macule usually found on the ____ or distal portions of extremities.

    <p>trunk</p> Signup and view all the answers

    Match the leprosy types with their characteristics:

    <p>Tuberculoid leprosy = Benign with few skin lesions Lepromatous leprosy = Severe with extensive skin lesions Borderline leprosy = Intermediate position Borderline tuberculoid leprosy = Unstable form with mixed features</p> Signup and view all the answers

    What is a common characteristic of lepromatous leprosy?

    <p>Slow progression and life-threatening if untreated</p> Signup and view all the answers

    ¾ of patients with leprosy present with multiple lesions that heal spontaneously.

    <p>False</p> Signup and view all the answers

    What is the role of genetic factors in leprosy?

    <p>Genetic factors contribute to susceptibility and response to infection.</p> Signup and view all the answers

    What is the usual concentration of sodium hydroxide (NaOH) used as a digestant and decontaminating agent?

    <p>2%</p> Signup and view all the answers

    The Ziehl-Neelsen method of staining requires the application of heat.

    <p>True</p> Signup and view all the answers

    Name one of the staining methods used for Acid Fast Bacilli (AFB).

    <p>Ziehl-Neelsen</p> Signup and view all the answers

    The optimal decontamination procedure requires an agent that is _____ and yields growth of mycobacteria.

    <p>mild</p> Signup and view all the answers

    Which staining method is more sensitive for detecting Acid Fast Bacilli?

    <p>Auramine-rhodamine stain</p> Signup and view all the answers

    Match the type of microscopy to its feature.

    <p>Standard light microscopy = Rapid diagnostic method Fluorescent microscopy = Sensitivity to AFB LED FM microscopes = Enhanced visibility of AFB</p> Signup and view all the answers

    Acid fast smears must be prepared directly from clinical specimens.

    <p>True</p> Signup and view all the answers

    What range percentage of bacterially contaminated mycobacterial cultures is considered acceptable?

    <p>2% to 5%</p> Signup and view all the answers

    What is one reason low sputum smear positivity is common in HIV patients with pulmonary tuberculosis (PTB)?

    <p>Poor immunity to localize the lesion</p> Signup and view all the answers

    GeneXpert can only detect TB in sputum samples.

    <p>False</p> Signup and view all the answers

    What is the common format of nucleic acid amplification test (NAAT) used for tuberculosis diagnosis?

    <p>PCR</p> Signup and view all the answers

    The __________ test is used to determine exposure to M. tuberculosis.

    <p>Tuberculin Skin</p> Signup and view all the answers

    Match the type of specimen with its volume for GeneXpert testing:

    <p>Sputum = 2-3 ml Urine = 30 ml Cerebrospinal fluid = 2-3 ml Tissue = 25-50 mg</p> Signup and view all the answers

    What is the purpose of the Quantiferon-TB Gold Assay?

    <p>To measure the CMI response to mycobacterial antigens</p> Signup and view all the answers

    Results from serological tests (IGRAS) are generally available within a week.

    <p>False</p> Signup and view all the answers

    GeneXpert testing results are processed within __________ hours.

    <p>2</p> Signup and view all the answers

    What does a result of '3+' indicate in the recommended scale by RNTCP?

    <blockquote> <p>10 AFB/oil immersion field</p> </blockquote> Signup and view all the answers

    Phenotypic methods are used solely for drug resistance testing in mycobacterium cultures.

    <p>False</p> Signup and view all the answers

    What is the general incubation time required for most pathogenic mycobacteria?

    <p>2-6 weeks</p> Signup and view all the answers

    The growth of Mycobacterium tuberculosis is enhanced by ________ CO2.

    <p>5%-10%</p> Signup and view all the answers

    Match the following culture media with their characteristics:

    <p>Lowenstein Jensen (LJ) = Egg based media with a shelf life of 1 year Middlebrook 7H10 = Serum albumin agar media with antimicrobial agents BACTEC 460TB = Automated radiometric method using Middlebrook 7H12 Liquid media = Allows more rapid growth of Mycobacteria</p> Signup and view all the answers

    What is a limitation of culturing Mycobacterium species?

    <p>They are slow growing and need 6-8 weeks for growing</p> Signup and view all the answers

    Which mycobacterium fails to grow on artificial media?

    <p>M. leprae</p> Signup and view all the answers

    Automated liquid culture methods can decrease recovery time compared to conventional methods.

    <p>True</p> Signup and view all the answers

    Study Notes

    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

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    Mycobacterium tuberculosis Lecture Dec. 9, 2023 PDF

    Description

    Test your knowledge on essential practices in mycobacteriology laboratories. This quiz covers protocols for handling tissue samples, identifying mycobacteria, and important equipment safety measures. Enhance your understanding of lab safety and techniques specific to mycobacterial disease.

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