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...

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, host, (MTB) is spread through and biochemical airborne droplets from properties. infected individuals. Infection occurs through inhalation. Lab Diagnosis Clinical Features Diagnosis involves Clinical features of microscopic examination tuberculosis include cough, of sputum, culture, fever, night sweats, and biochemical tests and 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 This unique cell wall also makes them bacteria that possess a unique cell wall, which gives them a difficult to treat and contributes to wall structure, making them resistant characteristic acid-fastness. their ability to persist in the to staining and disinfection. environment and within host cells.  Classification of Mycobacteria Mycobacterium tuberculosis Atypical Mycobacteria Complex (MTC) This complex includes several species, Also known as non-tuberculous including M. tuberculosis, M. bovis, and mycobacteria (NTM), these are a diverse M. africanum. group. 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/ Free-Living Obligate Pathogens Opportunistic Saprophytes Pathogens These species These species can exist These species primarily exist in exclusively thrive independently but become the environment, often found in pathogenic under specific in a living host, soil, water, or decaying matter. conditions, often when the host's relying on the They don't typically infect immune system is weakened. host's resources for humans unless introduced into M. avium complex, a group of survival. the body through a wound or closely related mycobacteria, is an 1. M. tuberculosis (MTB) other1.means. M. smegmatis example of an opportunistic 2. M. leprae pathogen. They can cause lung 2. M. gordonae disease, especially in people with weakened immune systems. Classification based on Tubercle BacilliHost Bovine Bacilli 1 Mycobacterium 2 Mycobacterium tuberculosis (MTB) bovis (M. bovis) is infects humans, a species primarily causing found in cattle. tuberculosis. This This bacterium is a bacterium is rare cause of responsible for the tuberculosis in majority of TB humans. 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.  Etiology M. tuberculosis M. bovis M. bovis BCG The primary causative A closely related species A live, attenuated strain of agent of tuberculosis, a that primarily infects cattle M. bovis used as a vaccine highly infectious and but can occasionally infect against tuberculosis, airborne bacterial disease. humans. particularly in children. These bacteria are acid-fast, meaning they retain the red stain in Ziehl-Neelsen staining, despite being decolorized with acid alcohol. M. tuberculosis Characters Morphology Growth Characteristics Cell Wall Composition M. tuberculosis is a slightly curved, M. tuberculosis multiplies slowly, with M. tuberculosis has a thick, waxy lipid rod-shaped bacillus. The bacilli are a generation time of approximately cell wall that provides resistance to about 0.2 - 0.5 microns in diameter 18-24 hours. This slow growth rate various environmental factors. The and 2 - 4 microns in length. They are contributes to the lengthy duration of cell wall plays a crucial role in the acid-fast, meaning they resist tuberculosis treatment. bacterium's virulence and survival decolorization by acidified alcohol within the host. after prolonged application of basic fuchsin dye. 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 Primary Infection Postprimary The initial infection with _M. Tuberculosis tuberculosis_ is known as a Subsequent disease in a primary infection. previously sensitized person, either from an exogenous source or by reactivation of a primary infection, is known as postprimary tuberculosis. Pathological Features Both primary and postprimary tuberculosis exhibit quite different pathological features. Differences between primary and postprimary tuberculosis Characteristics Primary Postprimary Site Lower lobe Apical region Local lesion Small Large Cavity formation Rare Frequent Lymphatic involvement Yes Minimal Infectivity Uncommon Usual Local spread Uncommon Frequent Primary tuberculosis is the first infection with Mycobacterium tuberculosis. Postprimary tuberculosis develops months or years after the primary infection. In postprimary tuberculosis, the patient is likely to be immunocompromised, and the infection becomes reactivated and spreads, often to the upper lobes of the lungs. 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 BreathFever and Night Chest Pain Cough is a common Shortness of breath is a Sweats Chest pain is a common symptom of tuberculosis. It common symptom of Fever and night sweats are symptom of tuberculosis. It can be dry or productive. It tuberculosis. It is caused by common symptoms of is caused by inflammation can be accompanied by damage to the lungs. tuberculosis. The fever is of the pleura, the lining of blood. Patients often report often low-grade and occurs the lungs. cough lasting more than 3 in the afternoon or evening. weeks. Clinical Illness with Tuberculosis The most common form of tuberculosis is pulmonary disease. Tuberculosis may affect the lungs, causing symptoms like hemoptysis (coughing up blood), chest pain, fever, and night sweats. Other common symptoms include anorexia (loss of appetite), weight loss, and fatigue. Tuberculosis can also cause cavity formation in the lungs, which can lead to further complications. Extrapulmonary Tuberculosis Spread of TB Commonly Affected Areas When Mycobacterium tuberculosis spreads beyond the Common sites include the genitourinary system, lungs, it can affect various organs and systems meninges, gastrointestinal tract, skin, lymph nodes, bone throughout the body. This disseminated form of TB is marrow, spine, and joints. called extrapulmonary TB. Extra pulmonary TB can present with various symptoms Tuberculosis bacteria can travel through the bloodstream, depending on the affected organ or system. reaching different parts of the body. Diagnosis of TB Infection 1 1. Non-Specific Tests 2 2. Radiological 3 3. Hypersensitivity Non-specific tests include Examination to TB Antigens elevated erythrocyte Chest X-ray is an important Tuberculin skin test (TST) and sedimentation rate (ESR) and diagnostic tool, revealing QuantiFERON-TB Gold (QFT) are complete blood count (CBC) characteristic patterns of lung commonly used to detect TB with monocytosis and involvement in TB infection. infection. neutrophilic leukocytosis. 4 4. Histopathological Examination 5 5. Bacteriological Examination Biopsy of lymph nodes reveals granulomatous Direct smear microscopy using Ziehl-Neelsen stain inflammation with caseation necrosis, a hallmark of 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 Specific less sensitive than other 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 sample to be positive. used to identify acid-fast bacilli (AFB) in a sample. AFB are characterized by their resistance to decolorization with acid alcohol. This resistance is due to the 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- Middlebrook Media BACTEC Media MGIT Jensen 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 detecting mycobacterial cultivating M. of mycobacteria. mycobacterial growth. growth based on tuberculosis. fluorescent detection of Contains a variety of Contains a defined The system measures the oxygen consumption. nutrients including nutrient composition, production of CO2 by glycerol, asparagine, and ensuring consistent growing mycobacteria The system is designed to malachite green to growth and facilitating the and reports the results in be safe and support the growth of use of selective agents a short period of time. environmentally friendly, mycobacteria. like pyrazinamide and reducing the risk of ethambutol. radioactive waste. Lowenstein-Jensen Egg Base Medium Lowenstein-Jensen (LJ) media is a solid culture medium used to grow mycobacteria. It is particularly useful for growing _M. tuberculosis_. LJ media is prepared using a base of coagulated whole eggs, potato flour, glycerol, and salts. It also contains malachite green as a selective agent, which inhibits the growth of most other bacteria. The resulting colonies are typically rough, non-pigmented, and grow slowly. LJ media is an essential tool for diagnosing tuberculosis. 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 Disadvan 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 T 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 media. tuberculosis directly in clinical samples, even before Probes are used to detect and identify specific DNA culture results are available. This technique is used to sequences, enabling the identification of Mycobacterium amplify specific DNA sequences, allowing for the rapid tuberculosis and other mycobacterial species. and sensitive detection of the pathogen. PCR also has 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. Smear Microscopy Smear microscopy is a rapid and cost-effective technique for detecting Mycobacterium in clinical samples. It involves preparing a thin smear of the sample on a glass slide and staining it with a specific dye to visualize the bacteria under a light microscope. Acid-Fast Staining Techniques Ziehl-Neelsen Stain Kinyoun Stain This is a classic technique This is a modified Ziehl- using carbolfuchsin dye, Neelsen method that uses a which stains the more concentrated dye mycobacterial cell wall. solution, eliminating the need for heat. Fluorochrome Staining This method utilizes fluorescent dyes, such as auramine- rhodamine, to stain mycobacteria for fluorescence microscopy. 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 growth. amino acids. weeks, sometimes from 35°C to 37°C. months, to achieve visible growth. Colony Morphology Tuberculosis (M. Avian (M. avium) Kansasii (M. kansasii) Fortuitum (M. tuberculosis) fortuitum) M. avium colonies are M. kansasii colonies are M. tuberculosis colonies rough, wrinkled, and buff- smooth, yellow, and M. fortuitum colonies are are typically smooth, colored. slightly raised. smooth, white, and cream-colored, and slightly raised. slightly raised. Biochemical Tests Biochemical Tests Catalase Test Nitrate Reduction Test Niacin Test These tests help Measures the production Determines the ability of Detects the presence of distinguish Mycobacterium of catalase, an enzyme Mycobacterium to reduce niacin, a metabolic species based on their that breaks down nitrates to nitrites. byproduct of enzymatic activities. hydrogen peroxide. Mycobacterium tuberculosis. Molecular Identification Target Gene Amplification Database Comparison Molecular identification involves using specific DNA sequences for identification. PCR is a key technique The sequenced DNA is compared to extensive for amplifying specific target genes, which are often databases of known mycobacterial sequences to highly conserved. identify the specific species or strain. 1 2 3 DNA Sequencing The amplified DNA sequences are then subjected to sequencing to determine the exact order of nucleotides. This provides a unique genetic fingerprint for identification. Polymerase Chain Reaction (PCR) DNA Amplification Target Genes PCR is a technique used to amplify specific DNA PCR primers are designed to target specific genes sequences, enabling the detection of Mycobacterium known to be present in Mycobacterium, allowing for species even in low concentrations. species-specific identification. Sensitivity and Specificity Rapid Results PCR offers high sensitivity and specificity, making it PCR provides rapid results, often within a few hours, a valuable tool for detecting Mycobacterium in enabling quicker diagnosis and treatment decisions. clinical samples. 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 Assays: Quantiferon-GOLD Blood Test 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 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. 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 This concludes the presentation on Mycobacterium Tuberculosis. We hope you enjoyed the information shared. For further questions or inquiries, please contact Dr. T.V. Rao MD.