Mycobacteria PDF

Mycobacteria Mycobacteria are a genus of bacteria that are characterized by their waxy cell walls. This unique feature gives them a distinctive appearance under a microscope and makes them resistant to many disinfectants. by Heba Rashed ILOs Classification of Mycobacteria Transmission and...

Mycobacteria Mycobacteria are a genus of bacteria that are characterized by their waxy cell walls. This unique feature gives them a distinctive appearance under a microscope and makes them resistant to many disinfectants. by Heba Rashed ILOs Classification of Mycobacteria Transmission and Pathogenesis Mycobacteria are classified Mycobacterium tuberculosis based on their habitat and host (MTB) is spread through airborne droplets from infected individuals. Infection occurs through inhalation. Lab Diagnosis Clinical Features Diagnosis involves Clinical features of microscopic examination of tuberculosis include cough, sputum, culture, indirect and fever, night sweats, and molecular methods. weight loss.  Definition of Mycobacteria Rod-shaped Bacteria Waxy Cell Wall Difficult to Treat Mycobacteria are a genus of aerobic They are known for their waxy cell wall, This unique cell wall also makes them bacteria that possess a unique cell wall which gives them a characteristic acid- difficult to treat and contributes to their structure, making them resistant to staining fastness. ability to persist in the environment and and disinfection. within host cells.  Classification of Mycobacteria Mycobacterium tuberculosis Complex (MTC) Atypical Mycobacteria This complex includes several species, Also known as non-tuberculous including M. tuberculosis, M. bovis, and mycobacteria (NTM), these are a diverse group. M. africanum. They are often found in the environment These species are responsible for and can cause infections in humans, tuberculosis in humans and animals. particularly in those with compromised immune systems. Non-Cultivable M. leprae is a unique species of mycobacteria that cannot be grown in artificial media. This makes it challenging to study and diagnose leprosy.  Classification based on Habitat Facultative/Opportunistic Obligate Pathogens Free-Living Saprophytes Pathogens These species These species can exist These species primarily exist in exclusively thrive in a independently but become the environment, often found in living host, relying on pathogenic under specific soil, water, or decaying matter. conditions, often when the host's the host's resources They don't typically infect immune system is weakened. for survival. humans unless introduced into M. avium complex, a group of closely the body through a wound or related mycobacteria, is an example 1. M. tuberculosis (MTB) other means. of an opportunistic pathogen. They 2. M. leprae can cause lung disease, especially in 1. M. smegmatis people with weakened immune 2. M. gordonae systems.  Classification based on Host 1 Tubercle Bacilli 2 Bovine Bacilli Mycobacterium Mycobacterium bovis (M. bovis) tuberculosis (MTB) infects is a species primarily found in humans, causing cattle. This bacterium is a rare tuberculosis. This cause of tuberculosis in humans. bacterium is responsible for the majority of TB cases worldwide. 3 Lepra Bacilli Mycobacterium leprae is the causative agent of leprosy, a chronic infectious disease that primarily affects the skin, peripheral nerves, upper respiratory tract, and eyes.  MYCOBACTERIUM TUBERCULOSIS COMPLEX The Mycobacterium tuberculosis complex is a group of closely related bacteria that are the primary cause of tuberculosis. Route of Transmission Mycobacterium tuberculosis is primarily transmitted through the air, specifically via droplet nuclei. These tiny droplets, measuring 1 to 5 micrometers in diameter, are released into the air when an infected individual coughs, sneezes, speaks, or sings. While droplet nuclei can remain suspended in the air for extended periods, they can also be generated during procedures involving infected lesions or clinical specimens. This poses a risk to healthcare professionals handling such materials. The inhalation of these droplets can lead to infection, particularly in the lungs. Pathogenesis Postprimary Tuberculosis Primary Infection The initial infection Subsequent disease in a previously with _M. sensitized person, tuberculosis_ is either from an exogenous source or known as a primary by reactivation of a infection. primary infection, is known as postprimary Pathological Features tuberculosis. Both primary and postprimary tuberculosis exhibit quite different pathological features. Clinical Features Tuberculosis (TB) is a disease that can affect different parts of the body. The majority of TB infections involve the lungs, known as pulmonary TB. However, TB can also affect other organs, known as extrapulmonary TB. Some common examples of extrapulmonary TB include TB of the lymph nodes, brain, kidneys, and bones. While less common than pulmonary TB, extrapulmonary TB can still be very serious. Symptoms and Signs of Tuberculosis Cough Shortness of Breath Fever and Night Sweats Chest Pain Cough is a common Shortness of breath is a Chest pain is a common symptom of common symptom of Fever and night sweats symptom of tuberculosis. It can be tuberculosis. It is caused are common symptoms tuberculosis. It is caused dry or productive. It can by damage to the lungs. of tuberculosis. The by inflammation of the be accompanied by fever is often low-grade pleura, the lining of the blood. Patients often and occurs in the lungs. report cough lasting afternoon or evening.  Extrapulmonary Tuberculosis Spread of TB Commonly Affected Areas When Mycobacterium tuberculosis Common sites include the genitourinary spreads beyond the lungs, it can affect system, meninges, gastrointestinal tract, various organs and systems throughout skin, lymph nodes, bone marrow, spine, the body. This disseminated form of TB is and joints. called extrapulmonary TB. Extra pulmonary TB can present with Tuberculosis bacteria can travel through various symptoms depending on the the bloodstream, reaching different parts affected organ or system. of the body. Diagnosis of TB Infection 1. Non-Specific Tests 2. Radiological Examination 3. Hypersensitivity to TB Examination Antigens Non-specific tests include Chest X-ray is an important Tuberculin skin test (TST) elevated erythrocyte diagnostic tool, revealing and QuantiFERON-TB Gold sedimentation rate (ESR) characteristic patterns of (QFT) are commonly used to and complete blood count lung involvement in TB detect TB infection. (CBC) with monocytosis and infection. neutrophilic leukocytosis. 4. Histopathological Examination 5. Bacteriological Examination Biopsy of lymph nodes reveals granulomatous Direct smear microscopy using Ziehl-Neelsen inflammation with caseation necrosis, a hallmark of stain and culture are essential for confirming TB infection. TB infection.  Bacteriologic Examination Sputum Blood Urine Biopsy Urine samples are useful A biopsy of affected Sputum is the most Blood cultures can help for diagnosing TB in tissue, such as lung common specimen for detect TB in cases of cases where the tissue, lymph nodes, or diagnosing pulmonary miliary TB or when the infection has spread to other organs, can be TB. It contains the infection has spread to the kidneys. examined for the bacteria responsible for the bloodstream. presence of TB bacteria. the infection. The type of specimen collected depends on the site of infection. For example, sputum is typically used for pulmonary TB, while biopsies and aspirates are used for extrapulmonary TB. Examination of Direct Smears Advantages Disadvantages Rapid The Ziehl-Nielsen stain is less sensitive than other Specific methods, such as culture, Inexpensive for detecting AFB. It Ziehl-Nielsen Stain requires at least 10,000 organisms per milliliter of The Ziehl-Nielsen stain is a specific stain used to identify acid- sample to be positive. fast bacilli (AFB) in a sample. AFB are characterized by their resistance to decolorization with acid alcohol. This resistance is due to the presence of mycolic acids in the cell walls of AFB. What is a significant limitation of the Ziehl-Nielsen stain when detecting acid-fast bacilli (AFB)? A. It is only used for pulmonary infections. B. It is non-specific and cannot accurately identify AFB. C. It is time-consuming and delays treatment. D. It requires a high concentration of organisms for a positive result. ANSWER: D Which staining technique uses carbolfuchsin dye to visualize mycobacteria? A. Gram Stain B. Ziehl-Neelsen Stain C. Kinyoun Stain D. Romanowsky Stain ANSWER: B Culture Advantages and Limitations Sensitivity Specificity Culture is highly sensitive, Culture is highly specific, detecting low bacterial loads. accurately identifying This helps diagnose infection Mycobacterium species. This even with limited bacilli. is crucial for accurate diagnosis and treatment. Time Consuming Culturing Mycobacterium takes several weeks. This can delay diagnosis and treatment, impacting patient care. Types of Media Lowenstein-Jensen Middlebrook Media BACTEC Media MGIT Media Available in both agar and Automated radiometric Automated non- Egg-based media is a broth forms, and is system provides rapid radiometric system for traditional method for designed for rapid growth detection of mycobacterial detecting mycobacterial cultivating M. tuberculosis. of mycobacteria. growth. growth based on fluorescent detection of Contains a variety of Contains a defined nutrient The system measures the oxygen consumption. nutrients including composition, ensuring production of CO2 by glycerol, asparagine, and consistent growth and growing mycobacteria and The system is designed to malachite green to support facilitating the use of reports the results in a be safe and the growth of selective agents like short period of time. environmentally friendly, mycobacteria. pyrazinamide and reducing the risk of ethambutol. radioactive waste. Middlebrook Media Middlebrook media is a type of culture medium used for the growth of mycobacteria. It is a solid medium that is based on an agar base. Middlebrook media is enriched with a variety of nutrients, including oleic acid, albumin, dextrose, catalase, glycerol, and malachite green. These nutrients provide the mycobacteria with the essential components they need to grow. Middlebrook media is often used in combination with other diagnostic tests, such as the Ziehl-Neelsen stain, to help confirm the diagnosis of tuberculosis. Middlebrook Media Advantages and Disadvantages Advantages Disadvantages Middlebrook media provides rapid results and enables the Middlebrook media is expensive, and not suitable for follow-up identification of the causative organism, which is important for studies due to its susceptibility to contamination. targeted treatment. The media also provides information about the sensitivity of the organism to different antibiotics. BACTEC MGIT (Mycobacteria Growth Indicator Tube) The BACTEC MGIT system is a rapid and automated method for detecting mycobacteria in clinical specimens. It utilizes a modified Middlebrook OADC medium containing oleic acid, albumin, dextrose, and catalase. The medium also includes a silicon film that acts as a fluorescence indicator. When viable mycobacteria are present in the tube, they consume oxygen, leading to a decrease in the fluorescent output of the sensor. The bottom of the tube fluoresces when exposed to ultraviolet light, indicating the presence of mycobacteria. The MGIT system can detect growth in as little as 5 days, making it a more rapid method compared to traditional culture techniques.  Molecular Techniques Molecular Probes (NAP) PCR (Polymerase Chain Reaction) This technique allows for the identification of PCR is a powerful tool for detecting Mycobacterium mycobacterial growth from both solid and liquid tuberculosis directly in clinical samples, even before media. Probes are used to detect and identify specific culture results are available. This technique is used to DNA sequences, enabling the identification of amplify specific DNA sequences, allowing for the rapid Mycobacterium tuberculosis and other mycobacterial and sensitive detection of the pathogen. PCR also has species. the capability to detect drug resistance mutations. Sample Collection and Handling Sterile Technique Use aseptic technique during collection to avoid contamination from other microorganisms. Appropriate Specimen Select specimen type based on suspected infection site, e.g., sputum, blood, urine, or tissue. Proper Labeling Label samples clearly with patient information and date of collection for accurate identification. Transport Media Use specialized transport media to maintain viability and prevent overgrowth of other organisms. Refrigeration Refrigerate samples promptly to preserve mycobacteria for optimal culture and testing. Cultivation of Mycobacterium Growing mycobacterium in a lab requires specific conditions and media. The goal is to create an environment that promotes their growth, allowing for identification and testing. Media Selection 1 Use selective media designed for mycobacteria, such as Löwenstein-Jensen (LJ) or Middlebrook 7H10. Incubation 2 Incubate at 35-37°C, with a 5-10% CO2 atmosphere. Growth Time 3 Mycobacteria grow slowly, often taking 2-8 weeks for visible colonies to form. Safety Precautions 4 Always handle mycobacterium cultures in a biosafety level 3 (BSL-3) laboratory to prevent exposure. The chosen media and incubation conditions are crucial for the successful cultivation of mycobacteria, ensuring proper growth for accurate identification and further analyses. Growth Requirements Temperature Oxygen Nutrients Time Mycobacteria are typically Most mycobacterial They require specific Due to their slow growth slow-growing organisms. species are aerobic, nutrients, including rates, cultures need to be They thrive at requiring oxygen for glycerol, fatty acids, and incubated for several temperatures ranging from growth. amino acids. weeks, sometimes months, 35°C to 37°C. to achieve visible growth.  M. tuberculosis Characters Morphology M. tuberculosis is a slightly curved, Growth Characteristics Cell Wall Composition rod-shaped bacillus. The bacilli are about 0.2 - 0.5 microns in diameter M. tuberculosis multiplies slowly, with M. tuberculosis has a thick, waxy lipid and 2 - 4 microns in length. They are a generation time of approximately 18- cell wall that provides resistance to acid-fast, meaning they resist 24 hours. This slow growth rate various environmental factors. The cell decolorization by acidified alcohol contributes to the lengthy duration of wall plays a crucial role in the after prolonged application of basic tuberculosis treatment. bacterium's virulence and survival fuchsin dye. within the host.  Non-Specific Tests Non-specific tests are used for screening of tuberculosis. They are not directly measuring Mycobacterium tuberculosis but measure the immune response to it. Tuberculin tests, such as the Mantoux test or PPD, are the most common non-specific test. They are based on the principle of delayed-type hypersensitivity. TST Interpretation The tuberculin skin test (TST) is a valuable diagnostic tool for tuberculosis infection. It assesses an individual's immune response to the Mycobacterium tuberculosis (Mtb) antigen. A positive TST reaction, indicated by an induration greater than 10mm, suggests exposure to Mtb. A negative TST, with an induration less than 10mm, suggests no prior Mtb infection. However, factors like BCG vaccination or allergies can influence the results. Tuberculin Skin Test Advantages The tuberculin skin test (TST) is easy to administer, safe for most individuals, and relatively inexpensive. It is a valuable tool for detecting latent tuberculosis infection (LTBI). Disadvantages The TST has limitations, including the need for multiple patient visits, low specificity due to cross-reactivity with environmental mycobacteria, and potential influence by prior BCG vaccination. Limitations The TST does not reliably differentiate between active TB and LTBI. It also has a high rate of false positives and false negatives. Therefore, it is not ideal for screening individuals in all situations. Interferon Gamma Release Release Assays: Quantiferon- Quantiferon- Blood Test GOLD Immune Response Quantiferon-GOLD is a blood It measures the immune test to detect latent TB response to specific TB infection. antigens in a blood sample. Sensitivity and Specificity Advantages Specificity Results within 24 hours It has high sensitivity and No need for a second visit specificity for detecting latent Less risk of false positives TB infection. Vaccine Bacille Calmette-Guérin (BCG) BCG is a live attenuated vaccine derived from a strain of Mycobacterium bovis. It is administered via intradermal injection in the deltoid. BCG is a compulsory vaccination for newborn infants. It helps prevent miliary and disseminated TB. BCG vaccination does not prevent TB infection. BCG is not recommended for individuals with an impaired cell-mediated immune response such as infants with primary immunodeficiency, HIV-infected individuals, burn patients, or patients with extensive, active skin disease.  The ______ method is a classic staining technique using carbolfuchsin dye. How long can the MGIT system take to detect mycobacterial growth? A. 5 days B. 1 day C. 3 days D. 10 days Smear microscopy requires a high concentration of mycobacteria to be effective. A. True B. False  Selected specimen types for suspected infection sites may include ______, blood, urine, or tissue. The culture method for Mycobacterium typically requires ______ weeks for cultivation. What is a primary advantage of using culture over direct smear examination? A. Culture can identify low bacterial loads effectively. B. Culture results are available almost instantly. C. Culture has no risk of contamination. D. Culture is inexpensive and requires no special equipment. CONTROL Tuberculosis control requires a multifaceted approach to prevent and treat infections. Key strategies include preventing transmission, early detection, and effective treatment. The WHO’s "End TB Strategy" aims to reduce TB deaths by 95% and incidence by 90% by 2035. Treatment of TB Treatment of tuberculosis is a long-term process that requires a combination of drugs. The treatment regimen aims to eliminate Mycobacterium tuberculosis from the body and prevent further transmission. The duration of treatment varies depending on the patient's condition and response to therapy. Treatment for active tuberculosis typically involves a multidrug regimen consisting of four primary drugs: isoniazid, rifampin, pyrazinamide, and ethambutol. These drugs work by targeting different metabolic pathways in the Mycobacterium tuberculosis bacteria. The effectiveness of treatment relies on the patient's adherence to the prescribed medication regimen. Poor adherence can lead to drug resistance and treatment failure. Thank You