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  • Comparing AFB and LED-Fluorescence Microscopy

    Content

    The comparison of the Acid-Fast Bacilli (AFB) and Light Emission Diode (LED) Fluorescence Microscopy is tabulated below:

     

    Direct Microscopy for Acid Fast Bacilli (AFB)

     

     

     

     

    Figure 1: Bright Field Microscope used in Ziehl–Neelsen (ZN) staining of AFB

    Light Emission Diode (LED) 

    Fluorescence Microscopy

    Figure 2: LED-Fluorescent Microscope used in fluorescent staining of AFB

    Figure 3: ZN-stained slide used in direct microscopy

    Figure 4: Auramine-O-stained slide used in fluorescent microscopy

    Method used widely for the diagnosis and confirmation of pulmonary tuberculosis

    Method used less commonly for the diagnosis and confirmation of pulmonary tuberculosis

    Staining procedure complex 

    Staining procedure simpler

    Examination at lower magnification is NOT possible

    Examination at lower magnification is possible

    Takes more time than LED-FM

    Takes 75% less time than ZN and chances to lose scanty slides are also minimized.

    Low durability

    High durability

    Less sensitive

    10% more sensitivity than Bright Field ZN microscopy 

    As specific as LED-FM

    Equally as specific as ZN microscopy 

    No need for an experienced lab technician

    Needs an experienced lab technician

    Technical errors are common

    Technical errors are less common

    Misses the paucibacillary TB cases, especially when the patient is co-infected with HIV

    Identifies the paucibacillary TB cases especially when the patient is co-infected with HIV

     

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  • Ideal Infrastructure Required for Microscopy Centers

    Content

    Under the National Tuberculosis Elimination Program (NTEP) network, the most peripheral laboratories are the Designated Microscopy Centres (DMC) which serve a population of around 100,000 each (50,000 population norm in tribal and hilly areas). 

     

    The basic requirements for microscopy centres include (Figure):

    1. Good ventilation by exhaust fan and open windows
    2. A table/ work bench to prepare smears
    3. A sink to stain smears
    4. A table/ bench to examine smears
    5. A table/ bench for documentation
    6. Basin for hand washing with supply of water
    7. Good lighting
    8. Non-slip flooring
    9. Sample receiving area

    Figure: Ideal Infrastructure required for Microscopy Centres

     

     

    Laboratory Diagnosis of Tuberculosis by Sputum Microscopy

    ESTABLISHMENT

    PROVISIONS UNDER NTEP FOR DMC FACILITIES 

    Infrastructure development

    Normative amount as per “Norms and basis of costing” for NTEP. Unit cost for initial establishment/ refurbishment/ upgradation/ maintenance of civil work to be carried out as per rates prescribed by Public Works Department (PWD) or cell/ division/ corporation/ wing for infrastructure development

    Human resources

    NTEP trained Lab Technician (LT)

    Diagnosis
    • Provision for sputum collection in the presence or absence of an LT
    • Adequate space for storing and using binocular microscope, essential lab consumables, space for recording and reporting
    • Availability of running water
    • Availability of electricity
    • Availability of biomedical waste management facilities

    Equipment

    Binocular microscope with a light source

    Location of DMC

    Ideally, the DMC should be located in a peripheral health facility that has at least 50 new adult OPD/day.

     

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  • Microscopy

    Content

    Microscopy is a TB diagnostic technology that utilizes the acid-fastness property of Mycobacterium tuberculosis to visualize it under a microscope. Results of sputum smear microscopy can either be smear-negative, or smear-positive (with various grades). 

    Advantages:

    • It is currently the most accessible and cheapest TB diagnostic test available under National TB Elimination Programme (NTEP) in India.
    • It has the shortest turnaround time for diagnosis.
    • It has high specificity. 

    Limitations:

    • Low sensitivity. It becomes positive only when more than 5000 bacilli/ml of sample are present. Hence, cases would be missed in early disease, or when an inappropriate biological specimen is provided, where bacterial load in sputum is less.
    • It is unable to differentiate between M. tuberculosis and Non-tuberculous Mycobacteria (NTM). This is predominantly an issue in geographies with lower burden.

    There are two types of microscopies used in NTEP: Ziehl-Neelsen (ZN) Microscopy and Fluorescence Microscopy (FM). These vary in the type of stain and microscope used. FM is newer of the two types and is currently recommended for use over ZN.

     

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