[Draft] Course for Lab Technicians (NAAT Lab) in NTEP

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Resources

• Global Tuberculosis Report, 2020; Geneva: World Health Organization, 2020
• Training Modules (1-4) for Programme Managers and Medical Officers India: Central TB Division, MoHFW, Government of India,July 2020

TB is caused due to the infection by a bacterium called Mycobacterium tuberculosis.

It often affects the lungs, and in such cases it is called Pulmonary Tuberculosis. But, it can affect almost any part of the body (except the hair and the nails), in which it is known as Extra-Pulmonary Tuberculosis.

Resources:

• Technical and Operational Guidelines for TB Control in India 2016

Tuberculosis is transmitted mainly through the air via droplet nuclei generated when a TB patient coughs or sneezes. 

It is estimated that every sputum smear-positive patient spreads the infection to 10 – 15 persons annually, if untreated..

Figure: Transmission of TB bacteria through air via droplet

Resources:

• Technical and Operational Guidelines for TB Control in India 2016
• WHO - Fact sheet details on Tuberculosis

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Following are the risk factors that increase the chances of developing TB disease in an individual:

Figure: Risk factors for developing active TB

Resources:

• Technical and Operational Guidelines for TB Control in India 2016

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• TB Infection (or previously known as Latent TB infection) is a stage in between uninfected and having active TB. In this stage the person has no symptoms and can only be identified using laboratory tests.

• The vast majority of infected people may never develop TB disease. However, to achieve TB elimination, it is important to treat TB infection in people at risk of developing active TB disease.

• It is a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens with no evidence of clinically manifested active TB.

• There is no single acceptable/reliable test for direct identification of Mycobacterium tuberculosis infection in humans. Tuberculin Skin Test (TST) and Interferon-gamma release assay (IGRA) are commonly used tests for identifying TB infection.

Resources:

• Latent Tuberculosis Infection Guideline

• Guideline for Programmatic Management of Tuberculosis Preventive Treatment in India

• Globally, an estimated 11 million people fell ill with TB (incidence) in 2021.
• Historically, it has been the top infectious disease killer. In 2021, there were an estimated 1.4 million TB deaths and an additional 187 000 deaths among HIV-positive people.
• Three countries accounted for 42% of global cases in 2021: India (26%), the Russian Federation (8.5%) and Pakistan (7.9%).

Image Figure: Estimated TB incidence in 2021, for countries with at least 100 000 incident cases; Source: Global TB Report, 2022.

Resources​

• Global tuberculosis report 2022.

TB is one of the top burdensome infectious diseases in India. It is estimated that, around 1/4th (26%) of the world's TB cases are in India, translating to about 30 Lakhs new TB cases emerging each year (TB incidence). Against this estimated incidence the National TB Elimination program reported around 19 lakh new and relapse cases in the year 2021.

An estimated 5 Lakhs deaths occur due to TB each year in the country, translating to about 1 case of TB death every one-two minutes. Compared to this, there are only about 60 thousand deaths due to HIV and about 77 deaths due to Malaria each year.

TB diagnosis and treatment services although provided free of cost in the public sector, the cost of accessing these services and related loss of wages drive the affected people with poverty (catastrophic costs). TB also has a huge impact on the world's and the country's economy because of loss of workdays (100 million workdays per year).

Assessment

Resources:

• ^*WHO Global TB Report 2021
• ^{^}Status of National AIDS Response
• ^$PIB MOHFW

Those who are suspected of having TB disease are first screened for symptoms like cough and fever for more than 2 weeks, blood stained sputum and weight-loss. If found positive on screening, then TB patients are referred for testing to the nearest health facility. If diagnosed with TB, then they are subsequently initiated on treatment. The TB patients initiated on treatment are regularly monitored with the help of field staff or digital interventions like 99DOTS and MERM (Medication Event Reminder Monitor) technology. NTEP staff also ensures that the TB patients are regularly followed up on monthly basis till their treatment completion.

Figure: Patient Flow

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The Government of India has committed to achieving the Sustainable Development Goals(SDG) targets related to ending TB by 2025 (5 years ahead of the global target).  This would mean that in 2025, the 2030 target of achieving 80% reduction in incidence, 90% reduction in deaths due to TB compared to that of 2015, is to be achieved.

Table: India's commitment to End TB by 2025.

Resources:

• National Strategic Plan (NSP) - 2017 - 2025
• Global TB report 2021
• END TB Strategy

The National Strategic Plan (NSP) for TB elimination 2017–25 is a bold strategic framework to drive the  acceleration of progress toward TB Elimination, and achieving the Sustainable Development Goal (SDG) and End TB targets for India. It expects to guide the activities of all stakeholders including the national and state governments, development partners, civil society organizations, international agencies, research institutions, private sector, and many others whose work is relevant to TB elimination in India. It is adopts strategies under four groups DETECT, TREAT, PREVENT, BUILD.

VISION: TB-Free India with zero deaths, disease and poverty due to tuberculosis
GOAL: To achieve a rapid decline in burden of TB, morbidity and mortality while working towards elimination of TB in India by 2025.

The results framework of the NSP outlines the various targets (impact and outcomes) to be achieved.

Resources

• Revised National Tuberculosis Control Program National Strategic Plan For Tuberculosis Elimination 2017–2025, Central TB Division, MoHFW, 2017

Assessment Questions

The National Tuberculosis Control Program (NTP) of India was launched in 1962. It relied on BCG, X-ray based diagnosis and Streptomycin and INH based treatment centralized at district level.  

Based on a review of the NTP, and WHO recommendations of the DOTS Strategy, Government of India then revised the NTP and launched new program with the title Revised National Tuberculosis Control Program (RNTCP) in 1997. It used Sputum microscopy at DMC(Designated Microscopy Centres) for diagnosis, and multi-drug Short Course Anti-TB Therapy,  decentralized to the TU (TB Unit) level. 

In recognition of the rising drug resistance problem the DOTS Plus/ PMDT (Programmatic Management of Drug Resistant TB) was launched in 2006 and scaled up to the entire country by 2012. 

Further to strengthen the monitoring and supervision system - a case based notification system - Nikshay was introduced in 2012. The same year Tuberculosis was added as a notifiable disease at the point of diagnosis by all health care providers.

Other key milestones from 2012 to 2020 were the availability of the Standards of TB Care in India (STCI) in 2014, introduction of the Daily weight band wise Fixed Dose combination (FDC) in 2016 and new drugs like Bedaquilline  and Delaminid were started in 2017 and 2018 respectively. 

To emphasise the commitment of the Government of India and to accelerate the efforts towards TB elimination, RNTCP was renamed as "National Tuberculosis Elimination Programme (NTEP)" in 2020.

Figure: Key milestones under NTEP

Resources:

• TBC India Website
• National Stratergic Plan for Tuberculosis Elimination 2017 - 2025

National Tuberculosis Elimination Programme (NTEP) is a centrally sponsored programme being implemented under the aegis of National Health Mission.

National Level: Managed by Central TB Division (CTD), the technical arm of the Ministry of Health and Family Welfare (MOHFW)

State Level: State TB Cell coordinates the overall TB elimination programme in state under the guidance of State Health Society. The training ,supervision, monitoring and evaluation NTEP at state level are looked after by STDC (State TB Training and Demonstration Centre).

District TB Centre (DTC) is the nodal point for all TB elimination activities in the district under the guidance of the District Health Society.

Tuberculosis Unit (TU) Level: NTEP activities at block/sub-district level are implemented through TU which comprises Designated Medical Officer (MO) supported by two full-time NTEP staff - STS (Senior Treatment Supervisor) & STLS (Senior TB Lab Supervisor).

PHI (Peripheral Health Institute): PHI is a health facility manned by a Medical Officer (MO). Some of the PHIs are also the Tuberculosis Diagnostic Centres, which are the most peripheral level laboratories in the NTEP structure. All the Private Health Facilities like Private Practitioners / Private Hospitals / Clinics / Nursing Homes are also PHI.

Figure: Organisational structure of NTEP

Resources:

• TB India Report 2021
• Technical and Operational Guidelines for TB Control in India 2016

TB is a notifiable disease in India, and TB notification has been made mandatory at the point of diagnosis since May 2012. This means that when a case of TB is diagnosed and/or put on treatment it is to be reported to the  NTEP.

• Every healthcare provider, i.e., clinical establishments run or managed by the Government (including local authorities), private or NGO sectors and/or individual practitioners, need to notify diagnosed or treated TB patient’s. 
• Reporting is to be done on the online reporting system called Nikshay and should include details of patient identification, and TB diagnosis.
• This, apart from enabling essential public health actions such as Treatment initiation, and Contact Tracing, chemoprophylaxis, but also enables provisions of Direct beneficiary transfer for Nikshay Poshan Yojana

Points to Note:

As per MCI code of ethics a registered medical practitioner giving incorrect information on his name and authority about notification amounts to misconduct and such a medical practitioner is liable for deregistration. It is the duty of the registered medical to divulge this information to the authorized notification official as regards communicable and notifiable diseases. 

Resources

• TB Notification Letter from GoI, 7 May 2012.
• TB Notification Amendment, 21 July 2015, MoHFW.

Nikshay is an Integrated ICT system for TB patient management and care in India. Nikshay was launched in 2012 and since then, various improvements have been made in the system.

Nikshay provides-

• A Unified interface for public and private sector health care providers
• Different types of Logins such as State, District, TU, PHI, Staff logins, Private providers, Chemist, Labs and PPSA/JEET Logins
• Integration of all adherence technologies such as 99DOTS and MERM
• Unified DSTB and DRTB data entry forms
• Mobile friendly website with mobile app

Nikshay is accessible either via web browser(https://Nikshay.in ) or mobile App called ‘Nikshay’ that can be downloaded from Google Play Store(Android).

Figure: Nikshay Login Pages

TB cases are generally classified on the basis of previous history of TB treatment into New and previously treated cases.

New case - A TB patient who has never had treatment for TB or has taken anti-TB drugs for less than one month is considered as a new case. 

Previously treated patients have received 1 month or more of anti-TB drugs in the past. They could be further classified as:

• Recurrent TB case - A TB patient previously declared as successfully treated (cured/treatment completed) and is subsequently found to be microbiologically confirmed TB case is a recurrent TB case. 
• Treatment After failure patients are those who have previously been treated for TB and whose treatment failed at the end of their most recent course of treatment.  
• Treatment after loss to follow-up - A TB patient previously treated for TB for 1 month or more and was declared lost to follow-up in their most recent course of treatment and subsequently found microbiologically confirmed TB case 
• Other previously treated patients are those who have previously been treated for TB but whose outcome after their most recent course of treatment is unknown or undocumented. 

Resources:

• Technical and Operational Guidelines for TB Control in India 2016

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What is the "continuum of TB Care"?

Tuberculosis in contrast to other infectious diseases affects humans over a long period of time. Stages such as uninfected, infected, and disease, do not have clearly demarcated time points and stages blend with each other when a person is converting or transitioning from one to the other. In addition, a person cured from TB may get re-infected and diseased, or still harbor a few dormant TB bacteria which may get reactivated at some future time; this too needs to be cared for. Hence, TB care is visualized as a continuous spectrum of care, with parts that requires varying type and intensity of services throughout a person's lifecycle, called the continuum of care.      

Continuum of TB care is a concept that emphasizes that care provision/ TB related health care services exists is in a continuum for an individual's lifetime. It includes services before and after the current episode of TB including vulnerable population, TB infection, post treatment follow-up and recurrence of TB. 

NOTE:

1. When interacting with a person it is important to locate the person in this continuum of care. This is done by a combination of screening for TB disease, testing for TB infection, asking for the previous history of TB/ checking for existing records in Nikshay using patient identifiers.
2. In alignment with the continuum of care concept, Nikshay follows a lifecycle approach. It is able to document and track a patient throughout the continuum using any of the Patient ID or Episode ID or any of the service identifiers(Test ID, Transfer ID etc). 

Resources

1. Guidelines for Programmatic Management of Tuberculosis Preventive Treatment in India.
2. Standards for TB Care in India.

Assessment

Active TB disease has 4 major symptoms (the 4 Symptom complex). Presence of any one of these symptoms without any other reason warrants evaluation for TB. These are:

Figure: Signs and Symptoms of TB

People affected with TB may experience other symptoms as well. These may be based on the site that is affected with TB or other more non-specific symptoms of an infection. The physician or doctor would evaluate these symptoms in view of diagnosis of TB.

Resources:

• Technical and Operational Guidelines for TB Control in India 2016

Screening for active tuberculosis (TB) a process to filter out people who are less likely to have TB, from a group. Screened positive people are likely to have TB and are confirmed subsequently using a TB diagnostic test. This will allow finite diagnostic testing resources to be used on the remaining.

Screening in TB may be performed ​using simple field tools (4 Symptom complex) and tests such as Chest X-ray, or a combination of both. ​Combination of both is the most effective, but is often not applied due to the practical difficulties in making a chest X-ray conveniently available.

Screening is an integral part of any general case finding effort. It is also applied systematically in specific situations.

1. At health care facilities (intensified case finding): Here those visiting are screened using the 4 symptom complex, often at the point of entry to the facility. Those screened positive may be fast-tracked to TB Diagnostic testing.
2. In vulnerable populations in active case finding efforts: Here the entire population identified for active case finding are screened using the pre-decided protocols by going door to door. 

Resources

• Systematic Screening for Active Tuberculosis; Principles and Recommendations, WHO 2013.
• National Strategic Plan for Tuberculosis Elimination 2020–2025.

National Tuberculosis Elimination Programme (NTEP) strives for all presumptive TB patients to be microbiologically confirmed. Under NTEP, the acceptable methods for microbiological diagnosis of TB are: 

Rapid diagnostic molecular test: Rapid molecular tests that use techniques like NAAT are very specific. They amplify the genomic material in the patient sample and hence enhances detection

• Nucleic Acid Amplification Test (NAAT) e.g., GeneXpert, TrueNat

  

  Figure: Genxpert Machine for CBNAAT

  

  Figure:  Truenat Machine

• Line Probe Assay

Nikshay being the final updated repository of information of TB patient services, different roles perform various actions on the patient and TB service information in Nikshay and keep it updated. These roles range from health volunteers and treatment supporters on the field to health providers and doctors at health facilities. Each role acts on or inputs information based on the services he/she provides. The information is required to updated when it is generated by the person generating it (eg by the CBNAAT LT once the results of a CBNAAT test is available). 

• Laboratory Technician (LT): This role encompasses LTs at all labs performing all types of TB related tests including those at District Microscopy Centre (DMCs), Cartridge Based Nucleic Acid Amplification Test (CBNAAT) and Culture and Drug Sensitivity Test (CDST) laboratories. The LT is responsible for  adding and updating test records (Test request and test result) in Nikshay that he/she performs. Nikshay in-turn provides the LT with the electronic register of tests performed at the PHI and also shares the updated information with other relevant stakeholders.
• Treatment Supporters and Health Volunteers: These field level volunteers (such as ASHAs) may enroll presumptive TB cases and refer them to the nearest PHI. Once a TB case is identified and linked to them for treatment support they can record and monitor TB patient adherence. Nikshay in-turn provides them with updated information of the patient and automatically calculates Treatment Supporters honorarium and enables its processing by the relevant authorities.
• Patients: Patients may view their updated TB health records including adherence information and status of DBT benefits processing in Nikshay through the TB Arogya Saathi Application. 
• Pharmacist/ Storekeeper: These ensure drug dispensation records of patients are updated along with related supply chain information in Nikshay and Nikshay Aushadhi.
• Health Staff: This is a group of roles posted to various PHIs(Peripheral Health Institutions) and their catchment geographies, ranging from CHOs and MPHWs to Medical officers of the PHI. They are responsible for ensuring that all records related to all patients in their catchment area, encompassing all functions from enrollment to post treatment follow-up. The Medical Officer/ Doctor in-charge apart from being the final accountable authority for ensuring updated and correct information is present in Nikshay, he/she needs to review and record treatment initiation (along with treatment regimen) and clinical decision in Nikshay with support from his/he health staff.
• Senior Treatment Supervisor (STS)/ Senior TB Lab Supervisor (STLS)/ TB Health Visitor: These roles train and support the above staff in ensuring that the information in Nikshay is up-to-date. They also review reports and coordinate feedback to the other staff to ensure optimum patient services.

The overall responsibility of ensuring completion of real-time updating of information/ data in Nikshay lies with the District TB Officer (DTO). The DTO is responsible for ensuring that the relevant staff are trained in the use of Nikshay.

Resources

• Training Modules for Programme Managers and Medical Officers.
• Direct Benefit Transfer Manual.

Assessment

Patient Identifiers are key for identifying a patient in the Nikshay. Ideally, there should be only one identifier for each patient which identifies patient as well as processes for lifetime. However, during the continuum of care, one identifier may not be appropriate to represent the episodes and other processes. Hence there are various ids which are tagged to one patient in Nikshay. Knowing each ID and its purpose is therefore important. The various IDs that are present in Nikshay and their purpose are described below:

1. Patient ID:  In Nikshay, a TB patient can be identified by their Name, Patient ID and Nikshay ID. Since, patients might get enrolled at one primary health institute (PHI), diagnosed at another and initiated on treatment at a different PHI, the ID has been simplified to a unique number. 
2. Episode ID: In a life cycle approach, a person needs to be tracked across episodes of TB known as the Episode ID. In the first episode, the Patient ID = Episode ID (will be numerically equal), the patient is notified and completes one treatment cycle (Diagnosis to outcome). However, the patient may continue to have TB or have TB again at a later point in time. This is recorded as a second notification and becomes the second Episode of TB. A patient may be identified in Nikshay using a global search by both Episode ID and Patient ID.
3. Test ID: When request for a laboratory test and test result for a patient is added under Nikshay, it generates a unique Test ID. For multiple tests added a new Test ID is generated for a patient.
4. Transfer ID: In Nikshay, a transfer request of patients between health facilities across the country feature is enabled. Users make requests of two types - “Transfer In” and “Transfer Out”. This process generates a unique Transfer ID for the patient. The details of Transfer In and Transfer Out with Transfer ID are available in Nikshay’s Transfer Management feature.
5. Sample ID: It is essential that patient samples are registered in Nikshay. While “Adding Test(s)” when sample details are added, Sample IDs are auto-generated. Sample IDs help to track samples in Nikshay using this unique ID.
6. Benefit ID: A “Benefit” defined in the Nikshay-PFMS (Public Finance Management System) is a payment due to a beneficiary under a particular scheme. For example, in Nikshay Poshan Yojana (TB Patient Nutritional Support Scheme), the beneficiary is a case of Tuberculosis, notified to Nikshay. This beneficiary under the scheme is eligible for Rs 500 for each treatment month. Thus Nikshay generates a Benefit ID that identifies the patient eligible for benefits @ Rs. 500 for each treatment month.
7. Beneficiary ID: A beneficiary is a person/ citizen who is eligible to get benefits (financial or in kind) under any government scheme. Whenever Nikshay identifies a potential beneficiary, it issues a unique beneficiary ID to it. All the benefits processed or paid to a beneficiary are tracked using the Nikshay Beneficiary ID. For example, if a patient has multiple episodes, all the benefits of the patient across episodes are managed using the Beneficiary ID. This information is available in the Beneficiary register exported from Nikshay.
8. Ayushman Bharat Health Account (ABHA) ID: The Ayushman Bharat Digital Mission (ABDM) aims to develop the backbone necessary to support the integrated digital health infrastructure of the country. Ayushman Bharat Health Account (ABHA) ID is a 14-digit number which can uniquely identify persons and authenticate them (previously known as Health ID). It can be used to access and digitally share one's health records, with consent. ABHA is integrated into Nikshay and addresses ABHA creation, capture and verification for seamless patient registration in Nikshay. Nikshay uses the Aadhaar verification services provided by NDHM (National Digital Health Mission) to generate ABHA.

Resources

• Nikshay-search for a patient, how to identify Nikshay ID/Patient ID, how to register a patient, Adding a new Episode, how to generate Test ID, Transfer Management, Sample ID generation_Diagnostics, Direct Benefit Transfer Manual, Training Video on Direct Benefit Transfer,  ABHA Workflow

Assessment:

Figure: Nikshay Home Page

After login in Nikshay, using the login credential shared by NTEP Health Staff, Treatment Supporter will be able to access the following button:

• New Enrolment: Allows to enrol new cases in Nikshay
• Search Patient: Allows to search for patients that are mapped to him /her, using Patient Name, Nikshay ID and Old Nikshay ID
• Add Patient Test: Allows to add tests for all the patients.
• Diagnosis Pending: View the list of the patients that are pending for diagnosis
• Not on Treatment: View the list of the patients that are diagnosed but pending to be initiated on treatment
• On Treatment Patients: Gives the list of on treatment patients
• Outcome assigned: Gives the list of the patients that have completed their treatment
• Training Material; Access the training content available on Nikshay
• Patient Summary: Gives a brief overview on the Presumptive cases registered, Diagnosed and patients that are initiated on treatment
• Task List: Allows to view the list of pending activities pertaining to adherence, Treatment Outcome and Bank details missing for mapped patient
• Latest Updates: New updates of features that are released on Nikshay

TB Aarogya Sathi empowers Citizens (including TB Patients under NTEP) and to serve as a Direct interface with the national TB program.

Citizen: The App is aimed at  increasing awareness among the citizens. It is available for all Citizens using the App (no login required to access this content)

• Information on TB (Symptoms, Side Effects)
• Health Facility Search
• BMI Assessment
• Nikshay Sampark Helpline
• Nutritional Advice

Patient: Patients registered with Nikshay will have access to the Adherence, Treatment Progress and DBT Details.

• Patients registered under Nikshay get access to their TB health record additional information (after login)
  • Adherence Details
  • Treatment Progress Details
  • DBT Details

TB Aarogya Sathi App is available in Google play store and can be download using this QR Code

Figure: TB Aarogya Sathi Application snapshot

Resources:

• Nikshay Training Material

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.

Resources

• WHO Policy Statement - Fluoresence Light-emitted Microscope for the Diagnosis of TB, 2010.

In Ziehl-Neelsen microscopy, the carbol fuchsin fuchsin stain is heated to enable the dye to penetrate and bind the waxy mycobacterial cell wall. Following acid-decolourisation, the sputum smear is counterstained with methylene blue which stains the background material, providing a contrast blue colour against which the red AFB can be seen.

On observation under a microscope with oil immersion at 100X magnification, AFB appears as red, straight, or slightly curved rods, occurring singly or in small groups, while the rest of the background, mucoid and pus cells are stained blue in colour.

The method was initially developed by Paul Ehrlich and later modified by afterwards the Franz Ziehl  and Friedrich Neelsen after whom the method is named.

Resources

• Laboratory Diagnosis by Sputum Smear Microscopy - The Handbook, GLI, 2013.
• Module for Laboratory Technicians, CTD, 2005.

Assessment

Fluorescence Microscopy is a newer and better type of microscopy where the TB bacteria are stained using a fluorescent dye using the property of Acid Fastness. The dye will fluoresce when illuminated by UV light. When the UV light source is an LED Lamp, it is called LED fluorescent microscopy.

Here the bacilli appear as slender bright yellow fluorescent rods, standing out clearly against a dark background, as can be seen in the figure below.

Figure: AFB as seen under an LED Fluorescence Microscope:

Principle of Fluorescence Microscopy

1. Cell walls of Acid-fast Bacilli (AFB) is made up of Mycolic Acid. The mycolic acid creates a waxy layer, making the cell wall impermeable to acids and alkalis.
2. The primary stain, a fluorescent dye called Auramine-O, binds to the cell wall of the bacilli.
   
3. Intense decolorization by acid alcohol does not release the primary stain. Thus, the AFB retain the colour of the primary stain, while other bacteria lose the stain
   
4. The counterstain, Potassium Permanganate provides a contrasting background and is useful to quench background fluorescence.
   

Advantages of LED-FM:

• Fluorescence LED microscopy is more sensitive (10%) than conventional ZN microscopy.
• It may be placed in existing DMCs and does not require any additional infrastructure.
• Examination of fluorochrome-stained smears takes less time.

Resources

• Manual for Sputum Smear Fluorescence Microscopy, RNTCP, MoHFW, 2007.

Cartridge Based Nucleic Acid Amplification Test (CBNAAT) is a rapid molecular diagnostic test. It is used for diagnosis of Tuberculosis (TB) and Rif-resistant Tuberculosis (RR-TB) in NTEP. Results are obtained from unprocessed sputum samples in about 2hours which helps in early detection and treatment of TB patients. 

India has vast number of CBNAAT laboratories which are utilized for TB/RR-TB detection and Universal Drug Susceptibility Testing (UDST) under the National TB Elimination Program (NTEP).  

Figure: CBNAAT Cartridge and Machine in Use (Image courtesy: USAID supported Challenge TB Project)

The CB-NAAT system detects DNA sequences specific for Mycobacterium tuberculosis complex and rifampicin resistance by Polymerase Chain Reaction (PCR). It concentrates Mycobacterium tuberculosis bacilli from sputum samples, isolates genomic material from the captured bacteria by sonication and subsequently amplifies the genomic DNA by PCR. The process identifies clinically relevant rifampicin resistance-inducing mutations in the RNA polymerase beta (rpoB) gene in the Mycobacterium tuberculosis genome in a real-time format using fluorescent probes called molecular beacons.

Video: Cartridge-Based Nucleic Acid Amplification Test [CBNAAT] - GeneXpert Technology 

Resources

• Training Module (1-4) for Program Managers and Medical Officers, NTEP, MoHFW, 2020.
• India TB Report 2021, National TB Elimination Program (NTEP), MoHFW, 2021.

Assessment Questions

Truenat is an indigenous rapid molecular test platform that is currently under use in NTEP for diagnosis of TB and Rif Resistance. It is a platform utilising real-time Polymerase Chain Reaction (PCR) technology built into micro-PCR chips.

Testing on Truenat involves three components:

1. Workstation (consisting of 2 devices)
   • Trueprep AUTO Universal Cartridge-based Sample Prep Device for the automated extraction and purification of DNA
   • Truelab Real-time micro PCR Analyzer for performing real-time PCR. It is available as 1 (Uno), 2 (Duo) or 4 (Quattro) chip ports.
2. Cartridge and Chip
3. Reagent kits (Sample Pre-treatment and Prep kits)

Figure: Truenat  Source: MolBio Products.

Test results for MTB detection and Rif Resistance has a turn around time of 1-2 hours. Depending on the micro-PCR chips used various tests can be performed using Truenat. Truenat MTB micro-PCR chips detect Mycobacterium tuberculosis bacteria for TB diagnosis. Truenat MTB RIF micro-PCR chip is used as a reflex test to detect resistance to Rifampicin (RIF), the first-line drug for TB treatment

Truenat has many advantages. Truenat is designed to be mobile and is battery operated (~8 hours on full charge). It can be deployed in peripheral laboratories and microscopy centres with minimal or no added facilities and hence it is more point-of-care. Biosafety requirements are similar to smear microscopy. However, it is multi staged and partially automated, requiring the presence of a Lab Technician through out the test.

Resources

1. Truenat MTB Kit Insert.
2. Trueprep AUTO Universal Cartridge-based Sample Prep Device.
3. Practical Guide to Implementation of Truenat Tests for the Detection of TB and Rifampicin-resistance, 2021.

Assessment

Line Probe Assay (LPA) is a rapid molecular test available at centralised laboratories.

The assay is based on Polymerase Chain Reaction (PCR) that can simultaneously detect Mycobacterium tuberculosis complex as well as drug sensitivity to anti-TB drugs.

Figure 1: The GenoType MTBDRplus Molecular LPA Procedure; Source: Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

Advantages of LPA

• Rapid molecular test. (Turnaround time: 3-5 days)
• Highly sensitive and specific.
• Performed directly from sputum smear-positive specimens and on isolates of M. tuberculosis complex grown from smear-negative and smear-positive specimens.
• Detects multiple gene mutations in anti-TB drugs.
  • First-line LPA detects mutations to rifampicin and isoniazid
  • Second-line LPA detects mutations to fluoroquinolones and aminoglycosides.
• Suitable for low and high-throughput labs.

Disadvantages of LPA

• Cannot be used as a point-of-care test.
• Requires appropriate laboratory infrastructure, equipment and biosafety precautions.
• Different rooms (DNA extraction, pre-amplification, amplification, post-amplification/ hybridization) are required to perform different steps (Figure 2).
• Requires trained manpower to perform tests and interpret test results.
• Stringent internal quality control is required to prevent contamination.

Figure 2: Amplification (A) and Post-amplification Laboratory (B) for LPA; Source: Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

Resources

• Guidelines for PMDT in India, 2021.
• Molecular Detection of Drug-resistant Tuberculosis by Line Probe Assay.

Assessment

Culturing TB Bacilli is well known and historic method for detection/ confirmation of Tuberculosis. It is a highly sensitive and specific phenotypic test; it can detect even a few viable bacilli in the sample (Upto 10 Colony Forming Units- CFUs). TB bacilli multiply in the culture and form colonies of TB bacilli which can are easily be identified.

Based on the growth media used Culture is divided in to two types, Solid and Liquid Culture methods. Types Culture:

• Solid Culture on Lowenstein Jensen media : Historic gold standard culture test. Results take usually upto 2 months (60 days).
• Modern Liquid culture systems: (e.g. BACTEC MGIT 960, BacT Alert or Versatrek etc.) Results take usually up to 42 days. 

Uses

1. Solid culture is the gold standard diagnostic test for TB. But it is not used for the purpose of TB diagnosis due to the long turn around time of 2 months. It is largely used for research purposes where it is used as the baseline test on which the sensitivity and specificity of other tests are calculated.
2. Liquid Culture is being used for follow-up monitoring of patients on drug resistant TB treatment to detect treatment failure. Liquid culture is also used for long term follow up patients who have successfully completed treatment to detect recurrence.
3. Liquid culture is used as a previous step to grow bacilli and obtain isolates prior to Drug Susceptibility Testing.
4. Liquid cultures are also used in TB prevalence surveys for its high sensitivity and specificity

Resources

• Guidelines for Programmatic Management of Drug Resistant Tuberculosis in India 2021

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Culture Drug Susceptibility Testing (CDST) is a growth-based phenotypic method used to check the susceptibility of Mycobacterium tuberculosis strains to various first and second line anti-TB drugs. Mycobacterial resistance to a particular drug is identified if there is growth observed in culture in presence of that drug.

In NTEP CDST is the standard method to detect resistance in samples of patients who have tested positive on followup. While CDST is possible on both Solid and Liquid culture, currently, the NTEP utilizes only liquid culture as a method for DST, due to faster Turn around times.

CDST testing services are available under NTEP in designated, specialized laboratories called CDST Labs both in public and private sector. Currently there are 80 such laboratories (60 certified for First Line and 49 for Second line drugs). Such designated laboratories are subject to regular external quality assessment, often by the National Reference Laboratory at that region.

Quality assured DST to R, H, Z, Mfx, Lfx, Lzd, Am, Km and Cm are available across the country. 

Resources

• Guidelines for Programmatic Management of Drug-resistant Tuberculosis in India, 2021.
• Training Manual for Mycobacterium tuberculosis Culture & Drug Susceptibility Testing, NTEP, 2009.
• RNTCP Laboratory Network Overview, CTD, 2009.

NTEP laboratory network is comprising of National Reference Laboratories (NRLs), state level Intermediate reference laboratories (IRLs), Culture & Drug Susceptibility Testing (C & DST) laboratories and peripheral level laboratories. Peripheral level laboratories consist of  designated microscopy centres (DMCs) and NAAT labs.

NTEP has a quality assured laboratory network for bacteriological examination of sputum in a 3-tiered system.

Figure: Laboratory network of NTEP

Resources:

• TB India Report 2021

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The National Reference Laboratories (NRLs) constitute the third tier of the National Tuberculosis Elimination Programme (NTEP) laboratory network hierarchy. 

They provide quality assurance and certification services for the Culture and Drug Susceptibility Testing (C&DST) labs and coordinate with the World Health Organisation (WHO) Supranational Reference Laboratory (SNRL) network.

There are six designated NRLs which are delineated in the figure below.

Image

Figure: Six National Reference Laboratories under NTEP in India

NIRT, Chennai, in addition to being one of the NRLs is also one of the WHO designated SNRLs for the Southeast Asia Region.

Resources

• Guidelines for Programmatic Management of Drug-resistant TB in India, 2021.
• NTEP Laboratory Network: Overview.

• National Reference Laboratories (NRLs) conducts annual on-site evaluation/supervisory visits to laboratories for assessing the quality of microscopy, culture and drug susceptibility test (C&DST), and for improvement of the overall laboratory quality. 
• NRLs also assist Central TB Division (CTD), in developing laboratory guidelines, standard operating procedures (SOPs), and conduct training to state-level Intermediate reference laboratories (IRLs) and other technical issues.
• NRLs conduct C&DST training to the IRLs, and develop SOPs for the technical procedures, equipment maintenance, infection control and recording and reporting. 
• NRLs are also responsible for offering second-line drug susceptibility tests (DST) for multi-drug resistant TB (MDR-TB) treatment failures. 
• NRLs are responsible for the accreditation of the mycobacteriology laboratory for culture and drug sensitivity testing under the National Tuberculosis Elimination Program (NTEP).
• In addition, NRLs are also responsible for the conduct of research for the programme and evaluation of newer tools for the diagnosis of TB.
• The National Institute for Research in Tuberculosis (NIRT) Chennai, the Supranational Reference Laboratory (SRL) of the region is responsible for the external quality assurance of the other 5 NRLs. NIRT is in turn quality-assured through the SRL coordinating laboratory at Antwerp, Belgium. 

Assessment Questions

Some Culture and Drug Susceptibility Test (C&DST) laboratories host an Intermediate Reference Laboratory (IRL) under the National TB Elimination Programme (NTEP). 

There is at least one IRL per state at an identified location, usually in a secondary or tertiary level public health facility. There are 34 IRLs in India.

The IRLs are responsible for:

• Undertaking training on laboratory technologies for district and field level staffs
• Conducting on-site evaluation visits to districts for sputum microscopy at least once a year
• Undertaking panel testing of Senior TB Laboratory Supervisors (STLS) at each district linked to it
• Ensuring the proficiency of staff performing National Tuberculosis Elimination Programme (NTEP) smear microscopy activities by providing training to laboratory technicians and STLS

Resources

• Guidelines for Programmatic Management of Drug-resistant TB in India, 2021.
• NTEP Laboratory Network: Overview.

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Under the National Tuberculosis Elimination Programme (NTEP), many labs are established at the regional level within states for providing Culture and Drug Susceptibility Testing (C&DST) facilities for presumptive TB/DRTB and for TB/DRTB patients. C&DST laboratories are mostly located in intermediate reference laboratories (IRLs) or medical colleges. There are 42 C&DST laboratories established under the programme in different geographies. Dedicated human resources are provided for the laboratories under the programme. Districts are linked with laboratories for providing facilities for Culture and DST using: Phenotypic Methods (Solid – Lowenstein Jensen (LJ), and Liquid Culture – Mycobacteria Growth Indicator Tube (MGIT)) Genotypic technology (Line Probe Assay (LPA) and Cartridge Based Nucleic Acid Amplification Test (CBNAAT))

Figure: Culture and Drug Susceptibility Testing (C&DST) facility, Source: The Foundation For Innovative New Diagnostics (FIND)

Resources

• Guidelines for Programmatic Management of Drug Resistant TB in India, 2021
• Training Modules (1-4) for Programme Managers and Medical Officers; New Delhi, India: Central TB Division, July 2020

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The National Tuberculosis (TB) Elimination Program (NTEP) has a network of Nucleic Acid Amplification Tests (NAAT) laboratories coupled with Designated Microscopy Centers (DMCs) to form the backbone of the diagnostic component of TB services.

Nucleic Acid Amplification Tests (NAAT) laboratories includes Cartridge-based NAAT (CBNAAT) and TrueNat tests. These tests detect tuberculosis as well as rifampicin resistance and are more sensitive than smear microscopy.

Functions of Nucleic Acid Amplification Test (NAAT) Laboratories:

1. Acting as a hub for collection of samples from public and private health facilities (spokes)
2. Universal Drug Susceptibility Testing (UDST) to rule out rifampicin resistance among confirmed TB patients
3. Timely provision of NAAT test result to the TB patient, medical officer of the concerned health facility and NTEP staff for related actions
4. Acting as a sample dispatch center for the Culture DST laboratory for subsequent processing of samples for first-line line probe assay (LPA) and second-line drug resistance testing utilizing second line LPA and liquid culture DST
5. Recording and reporting including digitization of diagnostic process from collection to test result in NTEP Nikshay portal and Laboratory Information Management System
6. Management of supplies and logistic associated with laboratory logistic (CBNAAT cartridges and TrueNAT chips) and reporting any additional requirement thereof
7. Supporting the quality assurance activities undertaken by District or Intermediate Reference Laboratory under NTEP
8. Support health system in carrying out special drives for vulnerable and at-risk population and their testing directly by CBNAAT (slum population, diabetic population, smoker, malnourished people, patients of silicosis and kidney dialysis etc.)

Resources

• RNTCP Technical and Operational Guidelines for TB Control in India, 2016.
• Guidelines for Programmatic Management of Drug Resistant Tuberculosis in India, 2021.

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Sputum microscopy diagnostic services under the National TB Elimination Programme (NTEP) are provided by the Designated Microscopy Centres (DMCs) established at the Peripheral Health Institution (PHI) level, where a functional binocular microscope and a trained Laboratory Technician (LT) is available. Light Emission Diode Fluorescent microscopes are provided to high-load PHI-DMCs such as that of the medical colleges. 

Based on latest directives, a DMCs may be established at all PHIs (Public and Private) of the country as needed. It is mandatory to have a DMC at all medical colleges in the country.

As molecular technologies like Truenat are also used in DMCs, NTEP has planned to rename DMCs as TB Diagnosis Centres (TDCs).

Criteria to be a DMC

The DMCs should satisfy the following criteria:

1. NTEP-trained Laboratory Technician (LT) should be present.
2. A functional binocular microscope should be present in the laboratory.
3. Physical infrastructure in the laboratory should meet NTEP guidelines.
4. Daily new adult outpatient cases of at least 60-100 and/or workload of at least 3-5 sputum smears per day for the LT in the laboratory.

DMCs in the public sector, at the onset of the programme, are provided with funds to undertake minor civil works to build up their physical infrastructure and are provided with binocular microscopes.

Human Resources Norms

• For the purpose of NTEP, a PHI is a health facility which is manned by at least a Medical Officer (MO).
• In addition to the MO and LT, there is 1 TB Health Visitor (TBHV) per one lakh urban population to support the urban TB control activities.

Other Criteria

Microscopy Centres may be established beyond population norms in medical colleges, corporate hospitals, Employee State Insurance Corporation (ESIC), railways, Non-government organisations (NGOs), private hospitals, Ayushman Bharat - Health and Wellness Centres (AB-HWCs), etc.

Before designating a DMC in other sectors, there should be a formal agreement by the hospital/ laboratory to take part in the External Quality Assurance (EQA) and to allow the concerned NTEP staff to supervise as per the NTEP guidelines.

If the above criteria are met by any private laboratory, the lab is considered for establishing a DMC.

• To provide better access for diagnosis of TB, all PHIs, wherever LTs and binocular microscopes are available, can be upgraded to a DMC irrespective of the population norms or OPD attendance.
• All DMCs should comply with the Quality Assurance (QA) mechanisms as per the EQA guidelines.

Resources

• NTEP Training Modules 1-4 for Programme Managers & Medical Officers, 2020.
• Operational Guidelines for TB Services at Ayushman Bharat Health and Wellness Centres.

Assessment Questions

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Functions and Integrated Services of the DMC

• Testing of Sputum samples by Microscopy.
• Request/ referral for microscopy or Nucleic Acid Amplification Test (NAAT) or Culture and Drug Susceptibility Test (C&DST) or Chest X-ray (CXR) or Tuberculin Skin Test (TST) is generated at the PHI-DMC, as well as follow-up tests.
• Maintain consumables and logistics required for testing/ packaging and transport.
• Maintain TB laboratory registers for recording and reporting.
• Notify every TB patient in Nikshay at the earliest and update information of patients on comorbidity, treatment adherence, treatment outcome, contact investigation and TB Preventive Treatment (TPT).
• Biomedical waste management for the waste generated at DMCs.
• A DMC is required to participate in the External Quality Assurance system(EQA) of NTEP to ensure standardized quality diagnostic testing. 

Resources

• NTEP Training Modules 1-4 for Programme Managers & Medical Officers, 2020

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To increase access to diagnostic services, NTEP has a provision for sputum collection centres in areas where the health facility is not equipped with key requirements to conduct sputum microscopy, molecular tests, drug susceptibility testing or follow up examinations.

Sputum collection centres are dedicated locations where sputum samples are collected, packaged and then transported to nearby TB diagnostic centres. It could be attached to any near-by health-facility as well.

Requirements of a Sputum Collection Centre

To function as sputum collection centres, the following is essential:

• Linkage/ mapping (time and distance) to testing laboratory
• Availability of adequate number of sputum cups and falcon tubes, logistics for sample packaging and transport
• Identification of open areas for sputum collection
• Staff trained in NTEP guidelines on sputum collection, sample packaging and transport, complete and correct documentation of laboratory request form, and infection control practices
• Feasibility and financial measures required for sample transport
• Inclusion of local volunteers, courier services, sample transportation under National Health Mission Free Diagnostic Services or other mechanisms as decided by the state/district
• Availability of Information, Education and Communication (IEC) material, training modules, and job-aids
   

 Sputum collection centres are established in:

• Ayushman Bharat Health and Wellness Centres/Sub-centres
• Urban primary health centres
• Tribal, hilly, desert and difficult-to-reach areas of the country

Resources

• Training Modules for Programme Managers and Medical Officers
• Operational Guidelines for TB Services at Ayushman Bharat Health and Wellness Centres
• Mycobacteriology Laboratory Manual, GLI Initiative, 2014

Assessment:

Presumptive TB case refers to a patient who presents with symptoms or signs suggestive of TB disease (previously known as a TB suspect) and where further diagnostic workup including bacteriological investigation is required.

 Presumptive TB can be categorized into

1. Presumptive Pulmonary TB (P TB) - Symptoms are directly related to lungs (Cough, hemoptysis)

2. Presumptive Extra Pulmonary TB (EP TB) - Symptoms/ signs are specific to an extra pulmonary site (example: Lymph node swelling)

3. Presumptive Pediatric TB - Symptoms of TB in young children are more difficult to identify and can be more general (fever, weight loss) 

Resources:

• Technical and Operational Guidelines for TB Control in India 2016
• Definitions and reporting framework for tuberculosis

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The National TB Elimination Program (NTEP), promotes the following principles to diagnose TB:

1. Try to establish the microbiological confirmation for all cases
2. Use rapid molecular diagnostics upfront wherever possible for diagnosis of TB and early identification of resistance to treating drugs.
3. Focus more on quality sample collection and timely transportation for a better microbiological confirmation 

Microbiological Confirmation for All Cases

Microbiologically confirmed TB refers to a presumptive TB case from whom a biological specimen is positive for acid fast bacilli smear microscopy, or positive for Mycobacterium tuberculosis on culture, or positive for TB through Rapid Diagnostic molecular tests - Nucleic Acid Amplification Test (NAAT) and Line Probe Assay (LPA). Establishing microbiological confirmation is key for all TB cases. Clinically diagnosing TB should be limited only to very few patients where, in-spite of high suspicion, microbiological confirmation could not be established, even after all possible efforts. The entire diagnostic algorithm puts utmost efforts to establish the microbiological evidence in a case of TB.

Upfront Rapid Molecular Diagnostics

Knowing the drug resistance pattern at the earliest is key for success of the treatment. Hence, the current policy highlights the importance of using molecular diagnostic test upfront wherever possible.

Complete diagnosis of TB is achieved by:

• Offering NAAT (CBNAAT/ Truenat) to all notified new patients and to test for resistance to Rifampicin. This is termed as Universal Drug Sensitivity Test (DST) for Rifampicin. Efforts are being made to collect specimen from all TB patients for NAAT at baseline.
• Testing individuals belonging to key population groups (clinically, socially vulnerable), those with extra pulmonary TB, people living with HIV and paediatric patients (after X-ray screening). They are directly referred for TB testing by NAAT
• For upfront NAAT, one specimen is tested using NAAT and if TB is detected, the other sample is used for further cascade testing by LPA and liquid culture

Quality Sample Collection and Transport

For TB diagnosis, it is essential that a good sputum sample is collected. A good specimen consists of recently discharged material from the bronchial tree with minimum amount of oral or nasopharyngeal material, presence of mucoid or mucopurulent material and should be 2-5 ml in volume. The specimen is collected in a sterile container after rinsing of the oral cavity with clean water. The collected specimens should be packaged and transported to the laboratory as soon as possible after collection.

Resources

• Guidelines for Programmatic Management of Tuberculosis Preventive Treatment, Central TB Division, MoHFW 2021
• Training Modules for Programme Managers and Medical Officers,Central TB Division, MoHFW 2020
• Guidelines on Airborne Infection Control, Directorate General of Health Services, MoHFW 2010

Assessment

For laboratory diagnosis of TB, different biological specimens are used.

Pulmonary TB: Sputum sample is used. Sputum is a thick fluid produced in the lungs and in the adjacent airways. Normally, a spot sample and a fresh morning sample is preferred for the bacteriological examination of sputum.

Extra Pulmonary TB:

Resources:

• Technical and Operational Guidelines for TB Control in India 2016

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The process for testing is initiated with a request for test and ends with the reporting of test results. In built into the process of testing is also the process of specimen collection and transportation.

1. Request for a test: This is the first step initiated when the requirement for a test is identified. This event is at the time of identification of presumptive TB/ DRTB, follow-up point (end of IP/CP, post treatment follow-up). The request may be initiated by the medical officer (MO) or the health staff at the Peripheral Health Institute (PHI) citing the reason for testing and the type of test required. The request is directed towards a laboratory where the required test is available. The request for test can be performed in Nikshay for any case that is already enrolled with an existing Patient ID. Requesting for test in Nikshay is analogous to filling up the physical request form Annexure 15A and generates a Test ID/ Test Request ID.

2. Patient Referral/ Sample collection and transportation: Following the request for test, the next step is to physically refer the patient to the corresponding laboratory, or collecting the appropriate biological sample and initiating its transportation to the lab. If biological sample is collected, the details of the sample need to be added under the request for test in Nikshay and the sample needs to be appropriately labelled and the corresponding details of the request test attached.

3. Performing the test: Once the sample has been received successfully at the destination laboratory, the lab technician (LT) updates the sample/ test record in Nikshay and initiates the relevant protocol for testing and follows through till results availability. 

4. Reporting results: Once the results are available it needs to be updated against the corresponding request for a test and it is visible to all relevant stakeholders in Nikshay. If only Annexure 15A is available, the results need to be updated there and needs to be manually communicated to the personnel initiating the request for test.

Resources

• Training Modules for Programme Managers and Medical Officers
• Guidelines for Programmatic Management of Tuberculosis Preventive Treatment

Assessment

Once a presumptive TB patient is identified, the patient is enrolled online by a healthcare worker or doctor in Nikshay online portal. For diagnosis of Tuberculosis, the treating physician can request a test utilizing the request test option of Nikshay online portal. The step-by-step approach for test request is as follows:

Step 0: Go to the Patients Page.

Step 1: Select the “Tests” tab.

Step 2: Click the “Add Test” button.

Step 3: Fill the form.

Step 4: Select the “Test Status” as “Results Pending”.

Step 5: Click the “Add Test” button by selecting the appropriate test for the patient.

In a situation where the patient is referred to another health facility for TB testing, one needs to select the test requested along with the facility name where the patient will visit for undergoing the TB test. the results are added by the concerned healthcare worker only after the test is conducted and the result is available.

In the absence of such results, it will show ‘Result pending’ instead of ‘Result available’ status.

Video: Process to add tests on Ni-kshay

Resources

• Nikshay Portal Training Resource Material.

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People who have been exposed to patients with infectious TB are known as TB contacts; they constitute a high-risk group for TB. Case finding investigation contributes to the early detection of TB cases, and results in identifying a significant number of additional patients.

Figure: Approaches to Tuberculosis Case Finding

Active case-finding requires systematic screening and clinical evaluation of populations who are at high risk of developing TB, such as people living in slums, tribal areas, congregate settings, persons who are household contacts of TB cases

Resources:

• Assessing TB Case-Finding

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Passive case finding is essentially where the patient self reports to the health care provider with symptoms. This requires that affected individuals are aware of their symptoms, have access to health facilities, and are evaluated by health workers or volunteers who recognise the symptoms of TB and link those individuals for TB testing services.

This approach to case finding has the least effort and cost and is a minimum expectation. In a Peripheral Health Institution (PHI), it is estimated that about 2-3% of new adult outpatients are symptomatic that require referral for TB diagnosis (presumptive TB cases).

Passive case finding may miss TB patients if :

1. The disease is mild/ transient.
2. Access to healthcare is poor.
3. Health providers do not have an adequate index of suspicion and are unable to reliably link respiratory symptoms to TB. 

Resources

1. Training Modules for Programme Managers and Medical Officers.

Assessment

Systematic screening of all individuals of a defined population is known as active case finding.  It is applied outside of health facilities at the community level by the health system.

Objective of ACF is to:

1. identify cases early, initiate prompt treatment, reduce risk of poor treatment outcomes and reduce risk of further transmission of TB
2. to provide access to diagnosis services to populations that would have been otherwise unreached

It is effort intensive and is recommended only in population groups where there is estimated high case load. In NTEP, ACF is recommended only to be performed in Key / vulnerable population.

ACF can also be clubbed with suitable ACSM campaigns to create awareness about the signs and symptoms and about TB in the target population/ community. It can also be combined with other health activities/ campaigns (such as Pulse Polio/ Leprosy screening/ population based screening for NCDs) for increased efficiency.

Resources

1. Training Modules for Programme Managers and Medical Officers.
2. Active TB Case Finding, Guidance Document.
3. WHO recommendations for Systematic Screening for Active Tuberculosis

Assessment

Intensified Case Finding (ICF) is a case finding approach between Active and Passive approaches. Here individuals coming in contact with the health system through any activity are screened actively for symptoms of TB and referred for testing.

This approach brings the benefit of active case finding approach by active screening for TB symptoms, but does limit the extensive effort required by restricting to only those people who has some or the other healthcare problem. This approach is considered for people attending a healthcare facility.

Some examples of ICF are screening for TB symptoms and referral for testing in:

• all cases attending an HIV clinic.
• among children with malnourishment who attend a nutrition clinic.
• all mothers attending the antenatal clinics

Resources

1. Technical and Operational Guidelines.
2. Assessing TB Case Finding.

Assessment

Bidirectional screening is a method to identify cases in diseases which have predisposition to each other or has a significant influence on each other. For example TB and HIV, where having HIV increases risk of developing TB and cases with TB would have poor outcomes if co-infected with HIV.

Screening for TB is done through four-symptoms complex based screening or through Chest X-ray. Screening for the linked disease is carried out as per the policies of the corresponding health program.

Bi-directional screening policies are implemented by various disease control programs. For example, with NTEP the following disease control efforts implement a bidirectional screening policy:

1. HIV through NACO 
2. COVID19 
3. Diabetes Mellitus (DM) through NPCDCS
4. Tobacco  through National Tobacco Control Program

Both programs monitor bidirectional screening, referral and testing as per their own policies.

Resources

1. National Strategic Plan for TB Elimination.

Assessment

The major duties and responsibilities of the Laboratory Technician at the Designated Microscopy Centre (DMC) are to: ​

1. Follow the standard operating procedures for sample collection, sputum smear microscopy​ and Nucleic Acid Amplification Test (NAAT)
2. Maintain the Tuberculosis (TB) Laboratory Register and early submission of the results to the medical officer managing the patient, which should also be updated in the Nikshay online portal in real-time
3. Coordinate with other staff to ensure that presumptive TB cases and symptomatic contacts of TB patients receive sputum containers with the necessary instructions to undergo sputum examination/NAAT
4. Assist the medical officer of the peripheral health institution (MO-PHI) in the identification of presumptive drug-resistant TB patients and ensure the collection and transportation of sputum specimens for NAAT/culture and drug susceptibility test according to the guidelines
5. Organize and supervise the disposal practices of contaminated lab material as detailed in the Laboratory Manual
6. Assist the Senior Tuberculosis Laboratory Supervisor (STLS) in the implementation of the National Tuberculosis Elimination Programme (NTEP) Lab Quality Assurance
7. Assist in the implementation of new TB diagnostic tools in NTEP
8. Facilitate change management with respect to use of Information and Communications Technology (ICT) and Nikshay tool for concerned data entry, validation, and its use for public health actions
9. Any other jobs assigned by the reporting officer

​

Resources

• DO letter - TOR and need norms for NTEP staff, 2021.
• Training Module (1-4) for Program Managers and Medical Officer, NTEP, MoHFW, 2020.

​

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The Medical Officer Designated Microscopy Centre (MO-DMC) at a Primary Health Facility is appointed from the General Health System. The MO-DMC is responsible for activities at DMC under the National TB Elimination Programme (NTEP).

Key Responsibilities of MO-DMC include

1. Screening and diagnosis

·        Clinical examination of all TB cases should be done by the MO. 

·        The MO should screen/refer:

o   All identified presumptive pulmonary TB cases for sputum smear microscopy, chest X-ray and presumptive Extra-pulmonary Tuberculosis (EPTB) cases for appropriate investigations

o   Presumptive TB cases with a negative sputum result to be referred for chest X-ray, followed by Cartridge-based Nucleic Acid Amplification Test (CBNAAT) as per diagnostic algorithm to ensure no TB case is missed

o   All diagnosed TB patients for Nucleic Acid Amplification Test (NAAT) for early diagnosis of resistance to Rifampicin (Rif)

o   All Rif-sensitive TB patients for first-line LPA testing

o   All presumptive TB patients for HIV testing

o   All diagnosed TB patients for HIV testing

o   HIV positive patients for TB by four symptom complex screening

o   All HIV positive TB patients to Antiretroviral Therapy (ART) centre for initiation of ART and Co-trimoxazole Prophylaxis Therapy (CPT).

2. Treatment initiation, follow up and treatment outcome

·        The MO should fill the original treatment card with details of treatment regimen according to weight-band and Drug Susceptibility Testing (DST) pattern.

·        It is the responsibility of the MO to ensure that all the diagnosed smear-positive patients start treatment or are referred for treatment.

o   All patients who are sensitive to Isoniazid (H) & Rifampicin (R) and all patients whose H & R status is not known should be initiated on first line anti-TB treatment.

·        The MO is responsible for clinically following-up the patient once in a month to:

o   Identify any ADR early

o   Assess clinical improvement

o   Support follow-up by laboratory investigations, whenever necessary

o   Control comorbid conditions like HIV and diabetes by appropriate treatment

o   Screen all patients for presence of symptoms of TB at the end of 6th, 12th, 18th and 24th month after completion of treatment and do a sputum culture in the presence of symptoms to diagnose recurrent TB.

3. Recording, reporting and TB notification

·        The NTEP Request Form for examination of biological specimens should be filled up completely by the MO.

·        The MO should coordinate with the STLS to ensure that tuberculosis-related laboratory services are properly performed and recorded by the laboratory technician.

·        Results recorded in the laboratory register, treatment cards and the TB Notification Register should be verified and ensured that they are consistent.

·        The MO should maintain TB Notification Register for patients diagnosed and transferred-in.

·        The MO should ensure that the treatment details are entered in Nikshay immediately.

·        Detailed description of symptoms and signs of ADRs to anti-TB drug should be recorded in TB Treatment Card by the MO.

·        The treatment outcome has to be recorded on the Treatment Card, Nikshay and the TB Notification register within one month of the event. Declaration of the treatment outcome has to be decided upon and signed with date by the MO.

·        The MO should ensure updating of Notification Register and Nikshay entry by the designated staff:

o   If any smear-positive patients are not entered in the TB Notification Register and are on treatment

o   For patients who have not been put on treatment after tracing them and putting them on treatment immediately

o   After collecting the bank account details of the patient for Direct Benefit Transfer of Nikshay Poshan Yojana.

4. Monitoring and supervision

·        Every week, the MO of the DMC should review the TB Laboratory Register to ensure that correct number of sputum smear examinations (two per presumptive TB case) are being performed for diagnosis.

·        The MO of the DMC should cross-check the results of the sputum examination in the TB Register with that of TB Laboratory Register and the TB Treatment Card.

·        The MO of the DMC should check the Tuberculosis Laboratory Register to make sure that all the columns have been completed.

o   The MO of DMC is responsible for determining the amount of reagents and consumables the DMC needs every month.

o   The MO should ensure uninterrupted supply of drugs; monitor monthly replenishment of stock to treatment supporter if drugs are not already
given and update in drug stock register and in Nikshay Aushadhi through designated staff.

·        The District TB Officer (DTO) conducts Random Blinded Rechecking (RBRC) of sputum smear microscopy and gives feedback and corrective actions to Lab technicians through MO-DMC.

Resources

Training Modules for Programme Managers and Medical Officers.

Assessment

Question

Answer 1

Answer 2

Answer 3

Answer 4

Correct answer

Correct explanation

Page id

Part of Pre-test

Part of Post-test

What are the key duties of a Medical Officer at DMC?

Screening and diagnosis

Advocacy and support to private practitioners

Treatment initiation, follow-up, treatment outcome

All the above

4

The Medical Officer (MO) at the DMC is responsible for screening, diagnosis, treatment initiation, follow-up, treatment outcome, monitoring and supervision, recording, reporting, TB notification, advocacy and support to private practitioners.

Yes

Yes

Peripheral Health Care Workers (PHWs) including Community Health Officer (CHO), Auxiliary Nurse Midwife (ANM) and Multi-purpose health worker (MPHW) are central to primary health care and service delivery. They play important an role in TB care at Peripheral Health Institutes (PHIs) and Designated Microscopy Centres (DMCs).

Their responsibilities include:

1. Vulnerable population mapping: vulnerability assessment and mapping of vulnerable population (diabetic patients, patients on immunosuppressants, alcoholics and smokers etc) in communities is done by PHWs.

2. Screening and referral for testing:

PHWs are involved in:

- screening of household/workplace contacts and other contacts of TB patients as eligible in the local context

- periodic active case finding among identified vulnerable population for TB/Latent TB Infection

- referring presumptive TB patients promptly to the nearest microscopy or molecular laboratory through laboratory request forms

- registration of referred cases in Nikshay as presumptive TB patient

- providing sputum container to persons with symptoms of TB and counselling for collection of good quality sputum in the morning

- sample packaging and transport to TB laboratories for testing

- follow-up sputum examination

CHO is responsible to ensure availability of adequate sputum collection containers (sputum cups and falcon tubes), logistics for sample packaging and transportation.

3. Treatment initiation: It is the responsibility of the PHWs in coordination with NTEP staff to organize and ensure treatment initiation for the patient. They decide upon a convenient location for drug administration, identification of treatment supporter and supply of drugs to treatment supporter

4. Coordinating treatment support:

- PHWs act as a treatment supporter or identify treatment supporter who is accessible and acceptable to the patient to provide TB treatment

- support for adherence to treatment and monitoring of TB patients at the community level

- update the original treatment card at the PHI on a fortnightly basis and Nikshay entries in coordination with NTEP staff

5. Ensuring public health action: 

The PHW visits the house of the patient within a week of TB diagnosis to:

- verify the residential address so that in case of interruption, retrieval action can be taken

- counsel the patient and family members regarding the disease, treatment and its adherence

- screening of contacts, providing TB Preventive Therapy (TPT) to all eligible

- advise patient on balanced diet, taking the food they can afford and also about nutritional support systems available for the eligible patients

- collect the bank account number of the patient or one of the household members; or facilitate getting the bank account opened, if not having one

- mobilize/refer for HIV testing

- sample collection and transportation for Dug Susceptibility Testing 

6. Awareness generation/advocacy in community

PHWs generate awareness/advocacy in the community. The activities include:

- awareness on health promotion and health seeking behaviour

- awareness on symptoms of TB, good cough etiquettes, available services for screening, diagnosis and treatment of TB

- awareness on patient support and benefit schemes including Nikshay Poshan Yojana

- mobilize community, community leaders (religious leaders, school principals, women’s Self-Help Groups, etc) and Panchayati Raj Institution (PRI) members for TB sensitization activities

- identify TB survivors to volunteer for the community engagement activities 

Resource

Training Modules for Programme Managers and Medical Officers, Central TB Division, MoHFW 2020

Assessment

The Senior TB Laboratory Supervisor (STLS) is the person responsible for monitoring the day-to-day activities of all the microscopy centres and nucleic acid amplification test (NAAT) sites and is thus essential to the success of the National TB Elimination Programme (NTEP). They also ensure the quality of TB diagnostic services.

Roles of STLS at the DMC

1. Program management

The STLS is responsible for ensuring that microscopy services in the district are well organized and the locations of the designated microscopy centres (DMCs) are known to all the medical officers in all peripheral health institutions (MO-PHI).

The STLS also ensures that there are:

• Uninterrupted staffing of DMCs, including coverage for laboratory technicians (LTs) that might be on leave, so that there is regular and uninterrupted availability of smear examination at the DMC.
• Uninterrupted supply of reagents and logistics required for the microscopy.
• Quality assurance and accuracy guarantee for the microscopic activities carried out.
• Regular training and continuing education of LTs.

The STLS reports to the district TB officer (DTO) in collaboration with the senior treatment supervisor (STS) regarding implementation, quality control (QC), supervision and management of laboratory supplies.

2. Monitor documentation related to microscopy

STLS ensures that all documentation related to sputum smear examinations is accurate and reports of examinations are given to the treating physician promptly. This includes:

• Each LT has a TB lab register which is filled completely and accurately.
• LTs understand the importance of limiting administrative errors (for example, keeping the sputum specimens with the proper lab forms for sputum examination and slides) and accurately recording the results of sputum smear examinations.
• LTs keep examined slides for review and on-site evaluation (OSE) visit by the STLS.
• There is an accurate recording of the results of the sputum smear examination.

STLS must explain to LTs that patients are diagnosed and placed on appropriate treatment regimens based on the smear results.

3. Ensure appropriate number and schedule of sputum examinations

• Presumptive TB persons should have their sputum examined the correct number of times for tubercle bacilli, at least 2 sputum samples should be examined.
• Follow-up cases should have 2 sputum samples examined and should be done according to the follow-up schedule.
   

4. Perform laboratory QC

This is done via OSE visits. The visit includes a comprehensive assessment of the laboratory safety including infection control measures; conditions of the equipment, adequacy of supplies as well as the technical components of acid-fast bacilli (AFB) smear microscopy employing a simple “Yes” and “No” checklist.

• The STLS visits every DMC under their supervision at least once every 4 weeks, and more often if possible.
• During these visits, at least 5 positive and 5 negative slides must be re-checked by the STLS.
• Staff at the DMC is supervised, evaluated and trained during these visits.
• The STLS maintains a diary, recording the details of these field visits.

At the end of each QC visit, detailed feedback is given by the STLS for continuous internal quality improvements. The STLS also ensures that centres maintain proper storage and transport of sputum specimens, the safety of lab staff and the maintenance of microscopes.

5. Waste disposal checks: STLS ensures that contaminated material is disposed of safely to ensure infection control. Sputum containers with sputum must either be incinerated, disinfected and autoclaved, or burnt in a pit and the burnt material buried.

6. Maintain an adequate supply of all materials necessary for microscopic examination

The STLS ensures that LTs have an adequate supply of reagents, sputum containers, slides and other materials including boxes for storing slides. This includes:

• Calculating the required volume of material (slides, sputum containers, etc.) required.
• Ordering supplies during the first week of the quarter to ensure uninterrupted supply at all DMCs.
• Distribution of sputum containers to all sputum collection centres/ DMCs in the area.
• Estimating and ensuring maintenance of adequate reserve stock at DMCs.

Resources

• NTEP Training Modules 1-4 for Programme Managers & Medical Officers, 2020.
• Module for Senior Tuberculosis Laboratory Supervisors, NTEP, 1999.
• Module for Laboratory Technicians, NTEP, 2005.

Assessment

At a DMC, the STS primarily ensures that 

1. All patient services from enrolment to outcomes for a TB patient are completed optimally. This is done by monitoring information submitted by the DMC such as referral for testing, no of people tested, no of people diagnosed with TB, initiated on treatment. 

2. All the patients started on treatment are tested promptly using the appropriate follow-up testing schedule( i.e. at the end of IP and CP). 

3. Maintain profile of the DMC in the Nikshay such as tagging the PHI as DMC, name of contact person and other particulars

4. Ensure data quality in the various records, both in physical and electronic records. This includes patient data, referral data and testing data.

Three things are received by the Lab Technician- Sample for testing, request form, and  request for test in Nikshay.

Accepting the request for testing includes the following steps:

1. The LT verifies details on the request form that has eleven parts.

• The first part contains details on the name of referring facility, name of the patient, complete address, age & gender of the patient, date of referral, type of presumptive TB, the key population to which the patient belongs and site of disease.
• The second part contains details of referring facilities, Nikshay ID, and the names of State, district and TB units.
• The third portion is for the diagnosis and follow-up of TB.
• The fourth portion is for the diagnosis and follow-up of drug-resistant TB.
• The fifth portion is to indicate the required tests with the details of the person requesting the test.
• Parts six to eleven are used for reporting test results.

2. The LT verifies the quality of the sample received.

3. LT captures details on the patient, reasons for testing, test requested, and the visual appearance of the sample in the TB Laboratory register.

4. LT verifies the test request generated in Nikshay against the test ID requested (Figure).

Figure: Test Details Added in Nikshay by the Referring Health Facility; Source: Guidelines for PMDT in India, 2021.

5. LT initiates the test requested.

Resources

• Guidelines for PMDT in India, NTEP, 2021.
• Training Modules (1-4) for Programme Managers and Medical Officers, NTEP, 2020.

Assessment

Presumptive pulmonary TB patients are subjected to sputum smear microscopy (Ziehl Neelsen (ZN)/ Florescence Microscopy (FM)). Two consecutive sputum specimens will identify the vast majority (95–98%) of smear-positive TB patients

Two specimens are collected:

• One Spot and one early morning sample OR
• Two supervised spot specimens collected at least one hour apart, and smears made from both the samples.

If one or both smears are positive, the patient is diagnosed as a microbiologically confirmed pulmonary TB case.

• The spot specimen collected is labelled as 'a'.
• While the patient is given a labelled container with instructions to cough out sputum into the container early in the morning after rinsing the mouth with water. This is the early morning specimen. This is labelled as specimen 'b'.

• If the health facility is not a Designated Microscopy Centre (DMC), then the patient is given a sputum container with the instructions to collect an early morning specimen and go with the sputum specimen to the DMC where the spot specimen can be collected.
• In case the patient is not able to travel to the DMC, then the spot specimen could be collected at the nearest health facility or sputum collection centre and transported to the DMC.
• These two samples should be collected within a day or two consecutive days.
• Two supervised spot samples may be collected one hour apart if patient is too sick, coming from a long distance or likelihood of not giving a second sample is significant.
• Collection of early morning specimens is preferred because of the overnight accumulation of secretions. However, spot samples collected at any time for patients is also suitable if productive sputum is expectorated after deep cough.

Resources

1. Module for Laboratory Technicians.
2. Training Modules for Programme Managers and Medical Officers.

Assessment

Educating patients on collection is essential to have good quality sputum. The healthcare worker (HCW)/ medical officer (MO) or the laboratory technician (LT) can educate patients on how to collect and dispense sputum.

The HCW/MO/LT provides a new sputum cup with the Laboratory Serial Number written on its side to the patient. They should explain that sputum should be collected in an open place or in a well-ventilated room; it should not be collected in closed rooms, toilets and ill-ventilated rooms

A specimen collected under supervision is likely to yield better results. Supervising person has to demonstrate how to collect good sputum and dispensing it:

1. Using a laboratory sputum cup, demonstrate how to open the lid of the specimen container and place it conveniently within their reach, so they can close it immediately after collecting sputum and also how to screw the cap on the cup tightly so it doesn't leak.
2. Demonstrate how to bring up sputum, beginning with rinsing their mouth as food particles may give false positive results.
3. Demonstrate deep inhalation (2–3 times) and let the patient know that this will initiate the cough reflex in most individuals.
4. Demonstrate how individuals can place their palms on the waist, squat or sit and continue deep breathing again. Tapping or thumping of the back 
   may encourage expectoration (Sitting and placing hands on the waist fixes the shoulder and pelvic muscles and brings the intercostal muscles of ribcage and diaphragm into action)
5. After deep inhalation and coughing deeply, the sputum should come up in their mouth. The sputum is retained in the mouth and allowed to fall from their tongue into the pre-labeled container. Patient should be encouraged not to spit into the container. The patient can also be encouraged to cough directly into the cup.
6. The patient’s mouth should not touch the container and the patient must ensure that sputum does not touch the outside of the container.
7. The patient should not open the sputum cup till they are ready to use it
8. They should not rub off the number written on the side of the container
9. They should not touch the inside of the container with their fingers or tongue while collecting sputum

The person collecting the specimen should make sure that no one stands in front of the individual who is trying to cough up the sputum. When an individual has only coughed up saliva or has not coughed up at least 2 ml of sputum, they should be encouraged to give good specimen that is of appropriate quantity.

Resources

• Module for Laboratory Technicians
• Training Modules for Programme Managers and Medical Officers

Assessment

The Healthcare Worker (HCW) needs to carefully explain how to collect a good quality sputum specimen. He/she needs to demonstrate how to bring up sputum from the chest, what a good sputum specimen looks like, and the quantity of sputum required.  

Characteristics of a Good Sputum Sample 

• Thick (semi-solid) muco-purulent (yellowish) in consistency, coughed out deeply from the lungs
• Sufficient in amount i.e., 2 to 5 ml (or enough to cover the size of a fingernail at the bottom of the container)
• It should not be blood-stained (brownish colour) as far as possible.

Steps to Ensure Good Quality Sputum Sample

1. Explain to the patient the characteristics of sputum - that it is thick and mucoid as compared to saliva which is thin and watery.
2. The patient should preferably rinse his/her mouth to get rid of any food particles which may give false-positive results.
3. One should demonstrate to the patient by action how s/he should take deep breaths and bring up the sputum.
   1. The patient is instructed to inhale deeply (2–3 times), which will initiate the cough reflex in most patients.
   2. The sputum is retained in the mouth and spit into the pre-labelled container without spilling.
4. Some patients may not be able to expectorate with deep breathing, in which case HCW should demonstrate to them how they should place their palms on the waist, squat or sit and continue deep breathing again.
   1. Tapping or thumping of the back may encourage expectoration. (Sitting and placing hands on the waist fixes the shoulder and pelvic muscles and brings the intercostal muscles of the ribcage and diaphragm into action).
5. When a patient has only coughed up saliva or has coughed up less than 2 ml (the size of a fingernail at the bottom of the container) of sputum, the patient should be encouraged to provide a better specimen.

Resources

• RNTCP Module for Laboratory Technician, CTD, MoHFW, 2005.

Biological specimen/ samples collected on reaching a TB testing laboratory needs to be formally received. The sample may be handed over by agents(couriers, health staff/ volunteers, patient representatives) or by patients themselves. The formal receipt of sample enables further processes such as testing and communication of results back to the patient. If the sample is successfully received, the appropriate testing process is initiated using the sample, else it is rejected and a fresh sample requested. 

The designated Lab Technician (LT) at the Laboratory is responsible for receiving the sample. To initiate the receipt, the sample along with the patient, or the sample along with accompanying test request is required. If it is available, following are the steps to complete sample receipt:

1. Compare patient information (Patient Name and Patient ID / Sample ID/ Test ID) to the test request that has been made.
   • This is performed by searching the patient ID in Nikshay under the Diagnostics menu and comparing and matching the patient details and the label on the sample.
   • If a physical form (Request for examination of Biological specimen for TB) is available, the details on the sample label and form needs to be compared and matched as well.
2. If the details are matched the LT then checks the quality (mucopurulent, non-blood stained), quantity (adequate to perform the requested test) of the sample and ensures that there is no leakage.
3. If the above checks are passed, then the LT Accepts sample. If not the LT rejects sample with a request to get a new sample. The rejection of the sample and request to obtain a new sample is recorded on Nikshay and is communicated to the patient and the person who requested the test.  
4. To register the receipt by accepting/Check in the sample for testing in Nikshay. The relevant information may also be captured in the TB Laboratory Register.  Ensure that the Test ID (if not already present) / Lab serial number from the Lab register is labelled on the sample container.

Sample receiving in Nikshay

Figure: Sample Journey Tracking in Nikshay; Source: Nikshay Diagnostic Training Content.

Resources

1. Training Modules (1-4) for Programme Managers and Medical Officers, NTEP, 2020.
2. Nikshay Zendesk, Nikshay Knowledge Base, FAQs.
3. Nikshay Zendesk, Nikshay Knowledge Base, Diagnostics.

Assessment

Storage conditions of sputum sample can effect the test results.

• Sputum samples should be transported to the laboratory as soon as possible after collection.
• It is the responsibility of Laboratory Technician (LT) and Senior TB Laboratory Supervisor (STLS) to ensure proper storage and transport of sputum specimens.
• Sputum is stored to preserve the specimen quality.
• The stored sputum samples should not be frozen.

Storage of Sputum Samples

For microscopy

• For smear microscopy, sputum specimens should be examined on the same day and not later than 2 days after collection.
• If delay is unavoidable, the sputum collected should be stored in a cool place/ refrigerated at 4°C to inhibit the growth of unwanted microorganisms.
• Stored sputum samples should be protected from light and heat to prevent liquefaction of the sample, else it makes the selection of mucopurulent part of the sample difficult.
• Samples received over holidays/weekends should be stored in a cool place/ refrigerated at 4°C.

For liquid culture

• Sputum should be stored in a cool place/ refrigerated at 4°C to inhibit the growth of unwanted microorganisms; not later than 3 days after collection.

For molecular tests - Nucleic Acid Amplification Test (NAAT) and Line Probe Assay (LPA)

• Sputum must be stored by refrigerating at 4°C to inhibit the growth of unwanted microorganisms and transported in cool chain to the nearest molecular laboratory. It should not be stored beyond one week at 4°C.

Resources

Technical and Operational Guidelines; Chapter 3: Case finding and diagnosis strategy

PMDT Guidelines 2021

Assessment

Sputum smears must be prepared promptly after samples are collected or received in the laboratory.

Steps in smear preparation are as follows:

1. Cleaning and Labelling of slide (No 1)
2. Making the smear (No 2-3)
3. Drying and Heat fixing the smear (No 4-5)
4. Staining and counterstaining the smear (No 6- 12)
5. Examination of slide/Reading the Smear (No 13-14)
6. Reporting and recording the observations (Digitally and TB Lab register)
7. Storage of slides (as per Laboratory Numbers in closed box) (No 15)

Figure: Steps in Smear Preparation; 1- Labelling of the slide, 2- Using a broomstick to pick up purulent portion (A) while avoiding the salivary portion (B), 3- Spreading sample on a glass slide, 4- Air-drying the slides, 5- Heat-fixing the smeared slides,6- Staining with 1% Carbol fuchsin, 7- Heating of stained smear, 8- Decolorize with 25% Sulphuric acid, 9- Rinse off decolourizer, 10- Counter stain with 0.1% Methylene blue, 11-Rinse off counter stain, 12-Drying the prepared slide, 13-14 Examination of smears , reporting of observations, 15-Storage of slides; Source: Laboratory Diagnosis by Sputum Smear Microscopy.

Resources

• Laboratory Diagnosis by Sputum Smear Microscopy - The Handbook, GLI, 2013.
• Module for Laboratory Technicians, CTD, 2005.

A Laboratory Serial Number is assigned to each presumptive TB patient who is examined at the microscopy centre.

Each Laboratory Technician (LT) needs to ensure that all the slides are labelled using the Laboratory Serial Number. This is essential for recording as well as for the review of the slide during the supervisory visit as well as during the quality assurance exercise.

For every test, a new slide needs to be used. It is essential that there are no fingerprints or any scratches on the side of the slide (see figure 1).

Figure 1: Always select new, clean, grease-free and unscratched slides

Once the LT is ready to prepare a smear, he/she needs to write the Laboratory Serial Number on the left side of the slide with a diamond marker or a grease pencil only (see figure 2). Avoid multiple labelling (see figure 3).

Figure 2: The laboratory serial number is written on one end of the slide using a diamond marker

Figure 3: Avoid Multiple Labelling; a grease pencil has been used to label the slide.

Labelling of slides needs to be monitored and supported by the concerned Senior TB Laboratory Supervisor (STLS) during External Quality Assessment (EQA) visits.

Resources

• RNTCP Module for Laboratory Technician, CTD, MoHFW, 2005.

Kindly provide your valuable feedback on the page to the link provided HERE

The National TB Elimination Programme (NTEP) has a standard procedure for sputum smear preparation, the steps for which are listed below:

The tools required for smear preparation include a clean work surface, new and clean glass slides, a discard bucket or a foot-operated bin with a plastic liner, bamboo or wooden applicator sticks or sterile wire loop, spirit lamp and a rack for drying smears.

• Use new, clean, unscratched glass slides and label the slide with the laboratory serial number. 
• Prepare the smear in the centre of the slide covering 3 cm X 2 cm

The smear is prepared by using either a wooden stick (Figure 2) or a sterile wire loop (Figure 3).

Steps for Smear Preparation Using a Wooden Stick

• Break the wooden stick into two halves with uneven ends.

• Using the uneven end, select and pick purulent portions of the sputum specimen and transfer onto a new, clean, labelled, glass slide.

• Using the wooden stick, spread the sputum evenly, in a continuous rotational movement, to cover two-thirds of the central portion of the slide. Smear preparation should be done near a flame. This is required as approximately 6 inches around the flame is considered as a sterile zone which coagulates the aerosols raised during smear preparation.
• Discard the used wooden sticks in the discard bucket or a foot-operated bin with a liner and disinfectant. A different broomstick is used for each smear so that one patient's sputum is not mixed with another patient's sputum.

​

• Air-dry the smear slide on the rack for 30 minutes
• After air-drying, heat-fix the smear, using a lighted spirit lamp.

Steps for Smear Preparation Using a Wire Loop

• Take a nichrome wire loop or a disposable loop.

• Using the loop, select and pick purulent portions of the sputum specimen and transfer onto a new, clean, labelled, glass slide.
• Using the loop, spread the sputum evenly, in a continuous rotational movement, to cover two-thirds of the central portion of the slide.

• After use, sterilize the loop in an electric loop sterilizer or flame the loop to red-hot.

​

• Air-dry the smear slide on the rack for 30 minutes.
• After air-drying, heat-fix the smear using a lighted spirit lamp.

Resources

• Module for Laboratory Technicians (RNTCP), Central TB Division, MoHFW, 2005.
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, GLI Initiative.

Kindly provide your valuable feedback on the page to the link provided HERE

For sputum smear microscopy, the slides should be air-dried as heating the slide while the smear is wet can result in bubbling of TB bacilli into the air.

Fixation makes the sputum stick to glass slide and preserves the shape of the bacilli. 

The procedure for air-drying and heat-fixing the slide is as follows:

• A smear prepared on a clean glass slide from mucopurulent portion of the specimen is air dried for 15-30 minutes on a rack (see Figure 1)

Figure 1: Rack for air-drying slides

• When dry, the smear facing upwards is fixed by heat from below. This can be achieved by passing the slide 2-3 times over the flame of a spirit lamp (as shown in Figures 2 and 3) for 3-4 seconds each time.

Figure 2: Fixing the Smear by Heat Fixation; Source: Laboratory Diagnosis by Sputum Smear Microscopy

Figure 3: Spirit lamp used to heat-fix smears

Important points to consider when fixing smears

• Heat fixing does not always kill Mycobacteria, exercise care when handling slides.
• Flame fixing may aerosolize bacilli from the smear.
• Overheating can damage the bacilli, burn the smear or break the slide.
• Insufficient heat or time can lead to smear washing off during staining steps.
• Heating for too short a period can result in a false-negative result because the TB bacilli will not be well preserved on the slide.

After the smears are fixed, they can be stained for examination or stored, or used in proficiency testing panel and quality control slides for staining.
 

Resources

Laboratory diagnosis by sputum smear microscopy

Method for Inactivating and Fixing Unstained Smear Preparations of Mycobacterium tuberculosis for Improved Laboratory Safety

Assessment

Good quality smears are essential for accurate examination and results. A good quality sputum smear is one that is of uniform thickness and made from the mucopurulent portion of the sample in the center of the slide (Figure 1).

Do’s and Don’ts of a Good Quality Smear

• Do ensure that the smear size is 3 cm by 2 cm
• Do ensure there are no fingerprints on the prepared smear

Figure 1: Uniform spread of smear, not too thick or thin, and covering an area of 3cm by 2cm

• The smear should not be very thick, but it should be thin enough to visualize a newsprint as can be seen in Figure 2

Figure 2: Smear thin enough to visualise a print through it

• All smears should be air-dried for 30 minutes, before heat fixing to ensure that the smear is not washed off during staining

Common Mistakes to Be Avoided

Resources

• The Handbook - Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, 2013
• Module for Laboratory Technicians – RNTCP, 2005
• Manual for Sputum Smear Fluorescence Microscopy- RNTCP

Kindly provide your valuable feedback on the page to the link provided HERE

After air drying and heat-fixing of the smear, it needs to be stained for the identification of MTB during microscopy.

The reagents required for the ZN staining procedure are shown in Figure 1. The steps involved in the staining procedure are shown in Figure 2.

Figure 1: Reagents Required for ZN Staining

Figure 2: Steps involved in ZN staining procedure

For a visual representation of the above-mentioned steps, please click the video below.

Resources

Laboratory Diagnosis by Sputum Smear Microscopy, GLI Initiative

Assessment

Characteristics of a Well-stained Slide

• Staining and appearance of Mycobacterium species in ZN staining.
  • The primary dye - carbol-fuschin stains all cells pink in sputum samples.
  • The bacilli retain the primary pink carbol-fuschin on decolourisation with acid-alcohol.
  • Epithelial, pus and mucous cells in the sputum decolourise on the addition of the acid-alcohol and take up the counterstain dye of methylene blue.
  • Thus, Mycobacterium species appears pink against a background of epithelial, pus and mucous cells that appear blue.
• ZN-stained Mycobacterium species are visible as pink, long, slender rods with granules or V-shaped or in clumps.
• Mycobacterium species should not be stained too dark or pale pink.
  • Possible reasons:
    • Smear thickness is not appropriate
    • Insufficient decolourisation
    • Low acid concentration
    • Carbol-fuchsin dries on smear
    • Primary staining/ decolourisation time not appropriate
    • Expired reagents used
  • Possible solutions:
    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time staining steps.
    • Do not overheat carol-fuchsin.
• The counter-stained cells should not be dark blue.
  • Possible reasons:
    • Smear thickness is not appropriate
    • Excessive counterstaining time
    • Inadequate washing after counterstaining
    • Methylene blue concentration is not appropriate
  • Possible solutions:
    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time staining steps
• There should not be any deposits on stained smears.
  • Possible reasons:
    • Stains not filtered
    • Slides not clean
  • Possible solutions:
    • Internal quality control of prepared stains
    • Filter stains
    • Use clean, fresh, unscratched slides for smear preparation.

Resources

1. Laboratory Diagnosis of Tuberculosis by Sputum Smear Microscopy - The Handbook, GLI, 2013.
2. Module for Laboratory Technicians, RNTCP, CTD, 2005.

Assessment

After air drying and heat-fixing of the smear, it needs to be stained.

Fluorescence microscopy (FM) staining should always be carried out in a designated area. The reagents required for the FM staining procedure are shown in Figure 1. The steps involved in the staining procedure are shown in Figure 2.

Figure 1: Reagents Required for FM Staining

Figure 2: Steps involved in FM staining procedure

For a visual representation of the above-mentioned steps, please click the video below:

Resources

Laboratory Diagnosis by Sputum Smear Microscopy, GLI Initiative

Assessment

Light-emitting Diode Fluorescence Microscopy (LED-FM) utilises the fluorescent dye Auramine-O to stain and detect Mycobacterium species in clinical samples.

Characteristics of a Well-stained Slide

• Auramine-O stained Mycobacterium species are visible as bright yellow/ green long slender rods, slightly curved, with variable lengths, single or in clumps, with uniform staining or granular appearance against a dark background (Figure A).

• Slides stained with Auramine-O should not have too much background fluorescence (Figure B)

      Possible reasons:

  • Smear thickness is not appropriate
  • Insufficient decolourisation
  • Counterstain too weak
  • Auramine-O dries on the smear

  • Auramine-O not filtered

    Possible solutions:

    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time-staining steps
    • Filter Auramine-O before use
    • Add a sufficient quantity of stains to cover the smear

• Slides stained with Auramine-O should not have pale fluorescence (Figure C)

      Possible reasons:

  • Smear thickness is not appropriate
  • Low Auramine-O concentration
  • Excessive decolourisation time

  • Stained smears exposed to daylight

    Possible solutions:

    • Internal quality control of prepared smears and stains
    • Use a stopwatch to time-staining steps
    • Store Auramine-O and stained slides in the dark
    • Read stained slides as early as possible
• Non-fluorescent yellow/ green coloured bacillary shapes should not be considered as Mycobacterium species.

• Slides stained with Auramine-O may contain stained artefacts/ background debris which are not Mycobacterium species.

   

Figure: Slides stained with Auramine-O showing bright yellow/ green slender rods (A); slide with too much background fluorescence (B); slide with pale fluorescence (C); Source: Laboratory Diagnosis by Sputum Smear Microscopy

Resources

1. Laboratory Diagnosis of Tuberculosis by Sputum Smear Microscopy - The Handbook, GLI, 2013.
2. Manual for Sputum Smear Fluorescence Microscopy, RNTCP, CTD.

Assessment

The manner and quality of smear reading has a major impact on the result of sputum smear microscopy and case detection. Each slide needs to be examined for at least 5 full minutes or 100 fields need to be examined. 

The overall process of reading a smear is outlined in the figure below:

Figure: Process of reading a smear

Resources:

• AFB Smear Microscopy, Trainer Notes.
• Module for Laboratory Technicians.
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy.

Assessment

The following are the measures to be kept in mind when adding immersion oil to the slide:

• Before adding immersion oil to the slide, ensure that the smear is facing upwards (Figure 1).

Figure 1: Slide should Face Upwards when Mounted on the Microscope Stage

• Add a drop of immersion oil, using the applicator (dropper bottle).
• The drop must fall freely onto the smear, so that the oil applicator does not touch the slide and get contaminated (Figure 2).

Figure 2: Oil Dropped onto Slide with an Applicator

• Mount the slide on the microscope and use the 10X objective lens to focus the smear, scan and look for mucoid material.
• Carefully rotate the 100X objective lens over the slide, adjust, sharp focus and observe under immersion oil.

Do’s and Don’ts:

• The 100X objective is the only lens requiring immersion oil.
• Keep the immersion oil away from other lenses.
• Use a good quality immersion oil.
• The immersion oil must have a medium viscosity and a refractive index (RI) greater than 1.5.
• Never allow the oil applicator to touch the slide.
• Do not use cedar wood oil diluted with xylene as it leaves a sticky residue on the lens and destroys the lens over time. 

Resources

• Guidelines for Laboratory Consumables, Central TB Division, 2019.
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, GLI Initiative.

Kindly provide your valuable feedback on the page to the link provided HERE

Image

Resources

• AFB Microscopy Trainer Notes
• Module for Laboratory Technicians
• Laboratory diagnosis by sputum smear microscopy

Kindly provide your valuable feedback on the page to the link provided HERE

Smears stained with the fluorescent dye, Auramine, are read with a Light Emitting Diode Fluorescence Microscope (LED-FM).

• The fluorescent-stained smears are to be read within 24 hours of staining in a dark room as the fluorescent stain fades on exposure to light.
• The slides are to be stored in the slide box to avoid exposure to light. Alternatively, they may be stored wrapped in brown or black paper and kept away from light.
• The slides should not be stored in a fridge as 4°C does not prevent or delay fading of fluorescent dye.

After fluorescent staining, smears are examined at much lower magnifications (typically 200x) than used for Ziehl Neelsen (ZN)-stained smears (1000x). Each field examined under fluorescence microscopy, therefore, has a larger area than that seen with bright field microscopy.

Examination Procedure

The steps include:

1. Switch on the LED-FM in a dark room.

2. Focus the stained slide using a 20x or 40x objective lens and view through the 10x eyepiece (magnification of 200x or 400x).

- Does not require the use of oil immersion.

3. Read smear in a linear pattern.

4. Count bacilli that appear as slender bright yellow fluorescent rods against a dark background in 30-50 fields. Rule out any artefact.

- For a trained and experienced Lab Technician (LT), each smear would take approximately 2 minutes for 30-50 fields or three horizontal sweeps.

Reporting Procedure

The smears are reported as negative/ scanty/ 1+/ 2+/ 3+ per the grading scale (Table).

Table: A Comparison between ZN (1000x) and FM (400x and 200x) for Grading of Smears at Different Magnifications

Abbr: AFB: Acid Fast Bacilli; HPF: High Power Field

Resources

• Training Module (1-4) for Programme Managers and Medical Officers, NTEP, MoHFW, 2020.
• Manual for Sputum Smear Fluorescence Microscopy, RNTCP, MoHFW, 2007.
• Fading of Auramine-stained Mycobacterial Smears and Implications for External Quality Assurance.

Assessment

Each Designated Microscopic Center (DMC) needs to maintain a National TB Elimination Program TB (NTEP) Laboratory Register which collects information of all presumptive TB patients who undergo tests or confirmed patients who undergo follow-up tests.

• After doing the Acid-fast Bacilli (AFB) test, the laboratory technician needs to ensure that all the microscopy test results are entered against the name and the serial number of the presumptive/ confirmed TB patients.
• The result needs to be mentioned in terms of positive or negative test results along with the AFB grading.
• It is expected that the positive results are written in RED and negative in BLUE point pens.
• The AFB results can be mentioned for newly diagnosed TB patients for both spot as well as morning sample and must include the Nikshay ID of the patient as well as the test date and result.
• In case, the sample is repeated, the same is also mentioned.
• All the information also needs to be entered into NTEP Nikshay online application using a tablet or mobile phone.
• In a setting where Nucleic Acid Amplification Test (NAAT) is co-located, another sample needs to be tested for Rifampicin (Rif)-resistance and the same result also needs to be noted into the TB laboratory register.
• The performance of each laboratory is also entered into the monthly DMC extract called Annexure M which is consolidated for all DMCs by the concerned Senior TB Laboratory Supervisor (STLS).
• HIV and diabetes tests are also offered to each confirmed TB case and the details of the same are also part of the TB laboratory register.
• Patients who have been tested also need to be provided with the test results in a hard copy apart from the provision of information to the treating clinician of the health facility.

Resources

• RNTCP Module for Laboratory Technician, CTD, MoHFW, 2005.

Kindly provide your valuable feedback on the page to the link provided HERE

Results of Ziehl–Neelsen (ZN) and Fluorescence Microscopy (FM) are added to Nikshay Diagnostic Module. After a patient is registered, test details are added which then leads to the page to add details on sample type, facility and test results (Figures 1-6).

Features of Nikshay Diagnostic Module

• A simplified user workflow for adding and updating test requests and results.
• Tests can be added and all the added tests’ history is available.
• Ability to map samples to one or more tests within Nikshay.
• A “Workbench” view is available to provide a single interface to view/ print/ copy the Test Summary, Add/ View Sample Details and Update/ View Result details.

Figure 1: Nikshay Screenshot to Add Tests under Diagnostics Module.

To add results for ZN/FM, the test types either Microscopy ZN or Microscopy Fluorescence should be selected from the drop-down menu (Figure 2).

Figure 2: Nikshay Screenshot to Add Reasons for Test under Diagnostics Module.

In case of follow-up with ZN/FM Microscopy, the reason for follow-up should be selected (Figure 3).

Figure 3: Nikshay Screenshot to add Follow-up ZN/FM Microscopy Test under Diagnostics Module.

Figure 4: Nikshay Screenshot to add Test Type for ZN/FM Microscopy and Facility Details under Diagnostics Module.

Details on sample type and sample description (mucopurulent/ blood-stained/ saliva) are added (Figure 5).

Figure 5: Nikshay Screenshot to add Sample Details and Description for ZN/FM Microscopy under Diagnostics Module.

Under test results, as shown in Figure 6, details of lab serial number, sample number, test date, test reported date and final interpretation of results are added based on the selection from a drop-down menu that includes options for:

• Negative
• Positive (1+, 2+, 3+)
• Positive (Scanty 1-9)

Figure 6: Nikshay Screenshot to add Test Results for ZN/FM Microscopy under Diagnostics Module.

Resources

• Nikshay Zendesk, Nikshay Knowledge Base, Diagnostics.

Assessment

The DMC lab register can be generated from Nikshay as a spreadsheet and stored electronically or printed for paper records.

Steps for generating DMC Register from Nikshay:  

1. Login to Nikshay Reports using your login ID and password.
2. Under Reports, click on ‘Patient wise List’ and select the option for ‘DMC Register’ (see screenshot below) 

Figure: Screenshot of Nikshay Reports to generate DMC Register 

A few filters need to be selected to generate the register.  

1. First is the geographic location (State/ District/ TU/PHI); most of these are preselected.
2. This is followed by a selection of the period for which the register has to be generated. This includes selection of ‘Frequency’ (either monthly or annually)of the report and the month or quarter for which the report is required. 
3. Click on the ‘Generate Excel’ button and it will lead you to a page from which you can download the register. 

Things to remember:

• You can generate a DMC register for facilities below your login level (e.g., a district can generate a DMC register of a TB Unit or DMC of the same district. and a State can generate a register for any District under it) 
• The data included in the register is based on the "Date reported" of a test.

Video: Steps to access the DMC Register in Ni-kshay

Resource 

Nikshay Reports Module 10 

Assessment 

The Tuberculosis (TB) Laboratory Register is a paper-based recording register kept in all National TB Elimination Programme (NTEP) laboratories for recording details of diagnostic services offered to TB patients referred from both private and public health facilities.

The register is maintained in the Designated Microscopy Centre (DMC). It is the only register used for recording the details of specimen smear examinations. The Laboratory Technician (LT) is responsible for maintaining and updating the laboratory register.

There are two portions in the TB lab register and the table below shows the details captured in each portion.

Important Points to Note

• Duplicate registers should not be maintained.
• LTs should ensure that the correct laboratory serial number is recorded.
• Laboratory serial number is given to the patient and not to the sample. A new number should be assigned to every presumptive TB case whose sputum is to be examined.
• All smear-positive (including scanty) results should be recorded in red ink.
• No over writing or manipulation in already entered data should be done.

The figure below shows the left-hand portion of the TB lab register. Click here to access the full form in the NTEP Training Module 2 for Programme Managers & Medical Officers, p. 219.

Figure: First Page of the TB Laboratory Register; Source: NTEP Training Modules 1-4 for Programme Managers & Medical Officers, p. 219

Resources

• Training Module (1-4) for Program Managers and Medical Officer, NTEP, MoHFW, 2020.

Kindly provide your valuable feedback on the page to the link provided HERE

Proper handling, maintenance and storage of the microscope are essential for proper functioning and life of the microscope.

The microscope should be placed and stored preferably in a box in a dry, dust-free and vibration-free environment, which is specially built in the laboratory (as shown in the figure below).

Figure: Storage of microscope in a wooden box; Source: Laboratory diagnosis by sputum smear microscopy

Important considerations for the storage of microscope

• When the microscope is not being used/ stored overnight, it should be covered or kept in a storage cupboard/box to keep it free from dust.
• Avoid exposing the microscope to direct sunlight.
• Avoid exposing the microscope to moisture as humidity may allow fungus to grow on the lens and cause rusting of the metal parts.
  • The stored microscope must be kept warm with a light source to prevent growth of fungus. A bulb holder for a 15-watt bulb should be fixed on the rear wall of the storage cupboard, such that the microscope does not contact the bulb while storing or removing the microscope. The bulb should remain on when the microscope is stored inside the box
  • An alternative may be to place plenty of dry blue silica gel into a shallow plate and place it in the box when the microscope is kept in it. Silica gel is blue in colour when it is dry, but when it becomes wet /absorbs moisture it turns pinkish. As soon as the silica gel becomes pink, it should be changed or heated until it turns blue again and then be reused.

Resources

1. Laboratory Diagnosis by Sputum Smear Microscopy.
2. Module for Laboratory Technicians.

Assessment

Regular cleaning of microscope is essential to ensure that it is optimally functioning. Cleaning is important to remove dirt, lens immersion oil and ensure disinfection. It involves cleaning the lenses, the body, the stage of the microscope and the light source. The microscope needs to be cleaned daily and the lenses need to be cleaned after each use.

Materials used for cleaning:

1. Lint free cloth
2. Lens paper
3. Lens cleaning solution
4. 70% ethanol
5. Detergent solution

Procedure for Cleaning Different Parts of the Microscope

1. The eyepiece shades, stage, focusing knob and nose piece are commonly touched during microscope operation, so these parts and the body of the microscope must be cleaned first to remove any stains with a neutral detergent /solution recommended by the manufacturer.
2. To clean the microscope eyepiece, objective lens, surfaces of the condenser and the light exit glass, moisten a lens paper with 1 to 2 drops of lens cleaning solution and clean the lens/ glass with a circular/spiral motion (as shown in the figure below).
   • Dry with a clean, dry piece of lens paper.
   • Use the Spiral Wiping Technique: Wipe from the center to the periphery in a circular motion.
   • The oil immersion lens should be cleaned after each use to ensure that immersion oil is not left on the surface.
3. The microscope surface and parts (except the lenses) need to be disinfected using 70% ethanol.

Figure: Cleaning of Lenses using the Spiral Wiping Technique. Source: How to Clean the Microscope

Important Considerations for Cleaning Microscopes

• Do not blow air to remove the dirt.
• Do not wipe the lens with an ordinary cloth. All the lenses should be cleaned with dry lens paper/lint-free cloth. 
• Never use spirit or alcohol or xylene to clean the lenses as these can damage them.
• Avoid using organic solvents that may damage plastic parts.
• Whenever possible, use the cleaning fluid recommended by the manufacturer.

Resources

• Laboratory Diagnosis of Tuberculosis by Sputum Smear Microscopy, GLI, 2013.
• Module for Laboratory Technicians, CTD, 2005.
• How to Clean and Sterilise Your Microscope, Olympus, Life Science Solutions, 2020.

Assessment

Removable parts in a microscope that need replacement include objectives, eyepieces, light bulbs, fuses. In case of repair/ technical support, only competent agency that handles maintenance of instruments should be contacted. Laboratory personnel should never attempt to dismantle any part of the microscope for repair.

Some of the common technical problems encountered, their likely causes and solutions are given in the table below.

In case, the viewing field is still dim and cloudy, consider the following possible causes and contact the competent personnel for cleaning/ repair:

• Massive growth of fungus on the lenses or prisms due to storage in a high-humidity environment
• Penetration of immersion oil between the lenses of the objective
• A damaged objective (as a result of careless focusing, dropping, rough changing of slides).

Resources

1.    

Module for Laboratory Technicians

2.    

Laboratory diagnosis by sputum smear microscopy

Assessment

The binocular microscope requires considerable care in its use, regular cleaning, and protection from dust and fungal growth.

Annual maintenance of a microscope includes:

1. Checking for broken or damaged parts and ensuring that the lenses, mirrors and other light-conducting surfaces are clean.
2. Cleaning the lenses first for dirt with a blower brush, then wiping with lens solution on a lens paper/ 70% alcohol on the lens paper.
3. Removing eyepieces or objectives from their fixation holes for cleaning.
4. Properly cleaning the lower lens of the condenser after removal from its fixing.
5. Cleaning the slide holder after removing from the mechanical stage.
6. Cleaning for any deposition of immersion oil and fungal growth on the prisms in the binocular tube and eyepieces
7. Changing electrical services including bulbs and fuses.
8. Greasing/ lubrication of movable parts.

National Tuberculosis Elimination Programme (NTEP) has earmarked INR 2000 for Annual Maintenance [AMC] per binocular microscope including spare parts and repairs.

Resources

1. Module for Laboratory Technicians.
2. Laboratory diagnosis by sputum smear microscopy.
3. Norms and Basis of Costing.

Assessment

• Designated Microscopy Centres (DMCs) are the most peripheral laboratory under the National TB Elimination Programme (NTEP) network.
• Any person identified to be a presumptive TB patient is first referred to the nearest DMC for sputum examination or their sputum specimens are collected and transported to the DMC. Therefore, it is very important for the DMCs to maintain an adequate stock of reagents and other consumables.
• The Medical Officer (MO) of DMC is responsible for determining the number of reagents and other materials the DMC needs every month.
• The Senior Tuberculosis Laboratory Supervisor (STLS) ensures these supplies are distributed in a timely manner, as and when required.
• The Lab Technician (LT) is responsible for exhausting the old supplies before the new ones.

The consumables required at the DMC can be broadly categorised as:

1. Consumables required for sputum collection
2. Consumables required for slide preparation
3. Consumables required for smear examination
4. Consumables stationery
5. Other

Resources

• Training Module (1-4) for Program Managers and Medical Officers, NTEP, MoHFW, 2020. 
• Guidelines for Quality Assurance of Smear Microscopy for Diagnosing Tuberculosis, RNTCP Lab Network, CTD, MoHFW, India, 2005. 
• Module for Laboratory Technicians, CTD, MoHFW, India, 2005.

Assessment

• Designated Microscopy Centres (DMCs) are the most peripheral laboratory under the National TB Elimination Programme (NTEP) network. Therefore, it is very important for the DMCs to maintain an adequate stock of all consumables.
• A paper-based stock register is maintained at the DMC and submitted as a part of the ‘Monthly report on programme management, logistics and microscopy’.
• All the DMCs as well as Peripheral Health Institute (PHI) that are a DMC need to fill the second part of this monthly report format for reporting the status of laboratory consumables and equipment (Figure below).

Figure: Monthly report format for reporting status of laboratory consumables and equipment, to be filled by PHI that is DMC.

• The stock register for consumables at the DMC has the provision to enter the information about the stock of consumables that are available at the DMC on the first day of the month, stock received and consumed during the month and stock remaining on the last day of the month along with the requested quantity of new stock.
• The Lab Technician (LT) of the DMC is responsible for exhausting the old supplies before the new ones.
• The Medical Officer (MO) of the DMC is responsible for determining the stocks and the Senior Tuberculosis Lab Supervisor (STLS) should ensure these supplies are distributed in a timely manner, as and when required.

Table: Calculation of Stocks Required at the DMC

Resources

• Training Modules (1-4) for Programme Managers and Medical Officers, CTD, MoHFW, GoI, 2020.
• Module for STS Part 2: Ensuring Proper Registration and Reporting. CTD, MoHFW, India.

Assessment

The Medical Officer (MO) of Designated Microscopy Centre (DMC) is responsible for determining the amount of reagents and other materials the DMC needs every month.

The Senior TB Laboratory Supervisor (STLS) will make sure these supplies are distributed in a timely manner, usually on a monthly basis or as and when required.

• STLS place an order for DMC reagents and consumable four times a year based on the approximate number of sputum specimens the laboratory examined in the previous quarter.
• The STLS should order the sputum containers during the first week of the quarter so that the health units and microscopy laboratories have enough sputum containers to collect sputum specimens and the microscopy laboratories have enough slides to conduct sputum smear examinations.

The calculation of reagents/ consumables required for examination of 3000 smears is shown in Table 1.

Table 1: Calculation of Consumables Required at DMC for Examination of 3000 Smears; Source: Training Modules for Programme Managers and Medical Officers

• The monthly report on Programme Management, Logistics and Microscopy at the Peripheral Health Institution (PHI) level are filled by DMCs which are PHI as shown in Table 2.

Laboratory Consumables (To be filled in by only PHIs which are a DMC)

* PHIs that are not a DMC, but have been supplied with sputum containers, should complete this row.

Table 2: Request for Laboratory Consumables and Reagents in monthly reports from PHI which are DMCs; Source: Monthly Report on Programme Management, Logistics and Microscopy Peripheral Health Institution Level

• The DMC reagents and consumables are budgeted in the District Annual Action Plan under laboratory materials as per the amount permissible based on District Performance in the last four quarters.
• Procurement planned during a financial year is based on last year’s specimen processed/ slides examined and amount spent for purchase of lab material for smear microscopy.

Resources

1. Training Modules for Programme Managers and Medical Officers.
2. Monthly Report on Programme Management, Logistics and Microscopy
   Peripheral Health Institution Level (https://tbcindia.gov.in/showfile.php?lid=3055)

Assessment

The National TB Elimination Program (NTEP) recommends freshly preparing reagents from commercial procured products, at the District TB units to be used for microscopy. Chemicals need to be carefully monitored for potency and the calculation needs to be accurate while preparing reagents for smear microscopy.

For Preparing 1% Carbol Fuchsin (500ml): The required chemicals and materials for preparing 500 ml Carbol Fuchsin are:

1. Basic Fuchsin

• Chemical name: Pararosaniline hydrochloride 
• Chemical structure: C19H18N3Cl
• Molecular Wt: 323.8
• Colour: Metallic green

Potency correction factor: Note down the dye content – this should be available on the container. The dye content should be approximately 85% - 88%. To calculate the required amount of basic fuchsin, divide the actual amount required by the dye content. For example: dye content = 85%, actual amount required = 5gms, required amount of dye = 5/0.85 = 5.88 gms.

2. Ethyl Alcohol: 50 ml (Absolute alcohol, purity must be 98-100%)

3. Carbolic acid crystals (Phenol): 25 gms

• Chemical name: Phenol
• Chemical structure: C6H5OH
• Molecular Wt: 94.11
• Melting point: 40°C+2.5

4. Distilled purified water: 500 ml

Steps for Preparing 1% Carbol Fuchsin (500ml)

1. Add 25 gms of phenolic crystals to a conical flask
2. Add 50ml ethanol
3. Mix well until the crystals completely dissolves. Add 50 ml distilled water if required.
4. Conical flask should be kept in water bath set at 60°C or in a trough containing warm water
5. Weigh required amount of basic fuchsin powder and transfer it into a conical flask
6. Mix well until the crystals dissolve well.
7. Make the total volume to 500ml by adding distilled water
8. Filter using Whattman filter paper and transfer into a bottle
9. Label as 1% Carbol Fuchsin - Primary stain
10. Date of preparation, date of expiry, batch no. and the name of the technician or STLS who has prepared the stain should be clearly mentioned on the bottle.
11. Store it in cool place, away from direct sunlight
12. Any time particles start to form in the Carbol Fuchsin solution, the solution must be filtered again

For Preparing 25% Sulphuric Acid (500ml):

• Chemical structure: H2SO4
• Molecular wt: 98.08
• Purity: 95-97%
• Colour: Clear

1. Measure 375ml distil water into a 1L flask
2. In a glass cylinder, measure 125ml concentrated Sulphuric Acid. Pour it slowly and gently into the conical flask containing distilled water. Note: Always add acid to water. Never add water to acid
3. To dissipate the heat generated, place the flask in a trough of water
4. Mix well
5. Allow to cool
6. Transfer into a bottle
7. Label as 25% Sulphuric Acid - Decolorizing Solution
8. Date of preparation, date of expiry, batch no. and the name of the technician or STLS who has prepared the solution should be clearly mentioned on the bottle.

For preparing 0.1% Methylene Blue (500ml):

• Chemical name: Methylthionine chloride
• Chemical structure: C16H18ClN3S
• Molecular Wt: 319.9

Potency correction factor: Note down the dye content – this should be available on the container. The dye content should be approximately 82%. To calculate the required amount of methylene blue, divide the actual amount by the dye content. For example: dye content = 82%, actual amount required = 0.5gms, required amount of dye = 0.5/0.82 = 0.61 gms.

• Weigh the required amount of Methylene Blue
• Dissolve it in 500ml distilled water
• Transfer into a bottle
• Label as 0.1% Methylene Blue
• Date of preparation, date of expiry, batch no. and the name of the person who prepared the solution should be clearly written on the bottle.

Always perform a quality check of the reagents prepared by using Quality Control Positive and Quality Control Negative. If purity is not available on the reagent bottle, then search the website of the company of which the reagent was procured or ask the company about the certificate mentioning about the purity of reagent

Please watch the video below for more information:

Resources

• Module for Laboratory Technicians (RNTCP), Central TB Division, MoHFW, 2005
• TB Lab Consumables Specifications 2019
• SoP for preparation of Laboratory Reagents - WHO
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, GLI Initiative

Kindly provide your valuable feedback on the page to the link provided HERE

Reagent preparation is an important activity and it’s essential to use certified chemicals and reagents. One should always check the potency of the chemicals used and calculate the amount to be weighed accordingly.

The following are the steps for reagent (primary stain and counter stain) preparation for Florescence Microscopy:

0.1% Auramine–O (1 L), the primary stain

• Weigh 1 gm Auramine–O and transfer to conical flask.
• Add 30 gm Phenolic crystals (99.5% purity) and mix well.
• Add 100 ml absolute Ethanol (98-100% purity) and mix well.
• Add 870 ml distilled water to make up final volume of 1 L.
• Filter and transfer to amber bottle.
• Label the bottle as 0.1% Auramine–O.
• Date of preparation, date of expiry, batch no. and name of the Senior TB Lab Supervisor (STLS) who has prepared the stain should be clearly mentioned on the bottle.

1% Acid Alcohol (1 L) to decolorize

• Dissolve 5 gm Sodium chloride in 250 ml distilled water.
• Add 5 ml concentrated hydrochloric acid.
• Mix with 750 ml absolute alcohol.
• Always add acid slowly to alcohol, not vice-versa.
• Store in amber coloured bottle.
• Label the bottle as 1% Acid Alcohol.
• Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

0.5% Potassium Permanganate, KMnO₄ (1 L), the counter stain

• Weigh 5 gm of Potassium permanganate (99.5% purity).
• Transfer to a conical flask.
• Take 1 L of distilled water.
• Add small amounts of distilled water tothe  conical flask containing KMnO₄ and mix well.
• Add the remaining distilled water to make up final volume of 1 L.
• Filter and transfer to a bottle.
• Label the bottle as 0.5% Potassium permanganate.
• Date of preparation, date of expiry, batch no. and name of the STLS who has prepared the stain should be clearly mentioned on the bottle.

Table: Preparation of Different Volumes of Stains for FM Microscopy

Resources

• TB Lab Consumables Specifications, 2019.
• SOP for Preparation of Laboratory Reagents - WHO.

Kindly provide your valuable feedback on the page to the link provided HERE

It is important to store slides, after reading as these will be required for Quality assurance - internal rechecking by the supervisor, and for External Quality Assurance (EQA) as per National TB Elimination Programme (NTEP) guidelines.

• Prior to storage slides need to be dried by gently placing the slide gently face down on the tissue paper or by wrapping it in the tissue paper and leaving it overnight to remove excess oil (Figure 1).

Figure 1: Using Tissue Paper to Drain Oil

• The slides are to be stored in the slide box in the same order as they are listed in the laboratory register (Figure 2).

Figure 2: Keeping the Slides in Same Order as Listed in the Laboratory Register

• One blank place must be left behind the first slide from a patient suspected with TB this allows the second slide of the same patient to be added after reading. This process will keep results consistent with the laboratory register (Figure 3).

Figure 3: Keeping Blank Spaces Between Slides for Consistency

• Always store slides in closed slide boxes away from sunlight (Figure 4).

Figure 4: Storing Slides in Closed Slide Boxes

Resources

• Manual for Sputum Smear Fluorescence Microscopy, RNTCP, MoHFW, 2007.
• Laboratory Diagnosis of Tuberculosis by Sputum Microscopy, GLI Initiative.

Kindly provide your valuable feedback on the page to the link provided HERE

False result: It’s a situation where the test result is different from the truth.

Types of False Results

• False Positive: When the test result is positive but the patient does not have TB.
• False Negative: When the test result is negative but the patient has TB.

Both cases will lead to wrong categorization and incorrect treatment. The consequences of false results are shown in the figure below:

Figure: Consequences of False Results in Sputum Smear Microscopy; Source: Quality Assurance of Sputum Microscopy in DOTS Programmes.

Resources

• RNTCP Module for Laboratory Technicians, CTD, MoHFW, 2005.
• Quality Assurance of Sputum Microscopy in DOTS Programmes Regional Guidelines for Countries in the Western Pacific, WHO, 2003.

Kindly provide your valuable feedback on the page to the link provided HERE

Quality Assurance (QA) is a systematic process used to determine the quality standards of TB laboratories.

Poor quality diagnosis may results in failure to diagnose TB or unnecessary treatment of non-TB case. Thus an effective QA mechanism is important for reliability of laboratory diagnosis.

QA in National Tuberculosis Elimination Programme (NTEP) consists of:

• Internal Quality Control (QC)
• External Quality Assessment (EQA)
• Continuous Quality Improvement (QI)

QC or Internal Quality Assurance is a systematic internal monitoring of working practices, technical procedures, checking equipment, new lot of reagents, smear preparation, grading etc

EQA is a process to assess laboratory performance by comparing laboratory results with a laboratory in higher tier i.e.Intermediate Reference Laboratory (IRL)/ National Reference Laboratory (NRL) through on-site evaluation, panel testing and random blinded rechecking of slides. Figure 1 depicts an eg of on-site evaluation with roles of various stakeholders and frequency of this activity. 

Figure 1: On-site evaluation (RNTCP Laboratory Network Guidelines)

QI is continuous monitoring of laboratory performance, finding non compliance and taking remedial measures to prevent recurrence of problems

Resources

• RNTCP Laboratory Network Guidelines.

Assessment

Sputum smear microscopy is an integral part of the National TB Elimination Programme's (NTEP’s) diagnostic services and can majorly impact the success of the programme.

It is essential to have a credible and well-established quality assurance system to assess the performance of the microscopy service.

NTEP has implemented a multi-level quality assurance network system of sputum smear microscopy in the country consisting of: 

1. Internal Quality Control (IQC)
2. External Quality Assessment (EQA)
3. Quality Improvement (QI)

• Internal Quality Control is a process of effective and systematic internal monitoring of the working practices.
  • It includes technical procedures, checking instruments, quality of new batches of staining solution, smear preparation, grading, equipment infection control measures, waste management, etc.
  • For the purpose of IQC, it should be ensured that all staining reagents’ dye content is mentioned on the bottle, concentrated acids are stored carefully in separate containers and stains are filtered before use.
  • Further, after each new batch of reagents is made, Quality Control Positive (QCP) and Quality Control Negative (QCN) slides should be prepared by the Senior TB Lab Supervisor (STLS) for quality control.
  • QCP slides should be prepared by pooling 3+ grade sputum samples, while QCN slides are to be prepared by pooling negative sputum samples with an adequate number of pus cells (≥10 pus cells/ field).
  • One set of QCP-QCN slides should be stained by STLS and another set should be given to the Designated Microscopic Centre (DMC) Lab Technician (LT) along with the reagent, and the results of both should be entered in the batch register/ IQC document.
  • All quality control slides should be stored for a maximum period of Four months. For the microscope, the lens should be cleaned with tissue paper after examining each slide and stored inside a microscope box at the end of the day.
  • The microscope box should contain silica gel and an electric bulb of 10-15 watts for desiccation to prevent fungal growth on the lens. The silica gel should be dehydrated periodically under direct sunlight. All microscopes should be covered under the Annual Maintenance Contract (AMC) with routine preventive maintenance.

• External Quality Assessment (EQA) is a process to assess the performance of the peripheral laboratories by a more competent laboratory, like the intermediate or the national reference laboratory. 
  • EQA has 3 components:

1) Onsite evaluation of peripheral laboratories by the supervisors from the reference laboratory under actual working conditions, in order to review the internal quality control mechanisms in place.

2) Panel testing of the slides (unstained and stained) received from the reference laboratory by the peripheral laboratory LT to evaluate the smear microscopy process. This helps to determine whether the LT can adequately perform Acid-fast Bacilli (AFB) smear microscopy.

3) Random blinded re-checking of a small sample of routine slides (both positive and negative) in the peripheral laboratory by the reference laboratory to assess the accuracy of the reading.

• Quality Improvement (QI) is a process by which all components of smear microscopy diagnostic services are continuously monitored and carefully analysed with the aim to identify the problems and implement corrective actions (including retraining) to prevent the recurrence of the problems.

Resources

• Training Module (1-4) for Program Managers and Medical Officers, NTEP, MoHFW, 2020.
• Guidelines for Quality Assurance of Smear Microscopy for Diagnosing Tuberculosis, RNTCP Lab Network, CTD, MoHFW, India, 2005.

Assessment

Panel testing is a method of External Quality Assurance (EQA) that is used to determine the adequacy of a laboratory technician to perform Acid-fast Bacillus (AFB) smear microscopy. This method evaluates individual performance in staining and reading and not the other laboratory activities.

• Panel testing under National TB Elimination Programme (NTEP) is used for Intermediate Reference Laboratories (IRLs) and District TB Centres (DTCs) during on-site evaluation, because these institutions do not have routine slides for blinded rechecking.
• A panel consists of a batch of stained and/or unstained smears that are sent out by the higher-level reference laboratory to the peripheral laboratories for processing, reading, and reporting of results
• Panel testing is not performed as a routine in DMCs, as they will have regular on-site evaluation and blinded rechecking.

Uses of Panel Testing

• Supplement re-checking programmes
• Provide information on the capabilities of the peripheral laboratories prior to
  implementing a re-checking program
• Assess status level of performance or to quickly detect problems associated with very poor performance
• Evaluate proficiency of laboratory technicians following training
• Monitor performance of individuals when adequate resources are not available to implement a re-checking program.

Resources

• RNTCP Laboratory Network Guidelines

Assessment

A field visit is an ideal way to obtain a realistic assessment of the conditions and skills practiced in the laboratory. Under National TB Elimination Programme (NTEP), On-site Evaluation (OSE) of Intermediate Reference Laboratories (IRLs) and District TB Centres (DTCs)/ Designated Microscopy Centres (DMCs) is therefore an essential component of a meaningful Quality Assurance (QA) programme.

OSE should be performed a tleast once a year by personnel from a higher-level laboratory (IRL/ National Reference Laboratory (NRL)) in order to evaluate the overall operational conditions in the microscopy centers.

Laboratory Supervisors must be knowledgeable in all operational and technical elements of AFB smear microscopy and have sufficient expertise to observe technicians performing routine tasks.

Importance of OSE

• Ensures that Standard Operating Procedures (SOPs) are in place and are displayed in all DMCs, internal Quality Check (QC) as per RNTCP is performed, and a functional binocular microscope is available
• Provides an opportunity for immediate problem-solving corrective action and on-site retraining.

Frequency of OSE (Figure)

• At least once a month visit by Senior TB Lab Supervisor (STLS) to the DMC, is required with re-checking of five positive and five negative slides.
• Regular on-site evaluation by District TB Officer (DTO) is important to assure recording and reporting of results; assessing operational conditions, safety, supplies, equipment and total workload.
• At least once a year visit by laboratory supervisors is recommended for IRLs by NRLs and for DTCs by IRLs.
• When poor performance has been identified through OSE, blinded rechecking or panel testing, additional visits by trained laboratory personnel from a higher-level laboratory (the IRL or NRL laboratory) supervisor are mandatory to perform a comprehensive evaluation of all laboratory procedures, implement corrective action, and provide training.

Components of OSE (Figure)

The visit includes:

• Comprehensive assessment of laboratory safety including infection control measures; conditions of equipment, adequacy of supplies as well as the technical components of Acid-fast Bacillus (AFB) smear microscopy. Sufficient time must be allotted for the visit to include observation of all the work associated with AFB smear microscopy, including preparing smears, staining and reading of smears.
• Examining a few stained positive and negative smears to observe the quality of smearing and staining as well as condition of the microscope
• Facilitating quality improvement through on the spot problem solving and suggestions for corrective action, wherever needed.

The NRLs provide training to all IRL personnel responsible for OSE. Additionally, non-laboratory personnel (e.g., DTOs) should acquire working knowledge of routine laboratory operations, including proper NTEP procedures, appropriate supplies, laboratory safety, basic microscope operations, and requirements of panel testing or rechecking programmes.

Figure: Frequency and Components of On-site Evaluation; Source: RNTCP Laboratory Network Guidelines

Checklist for IRL OSE and NRL OSE

On-site quarterly evaluation report of DTCs visited by IRL team to be given to concerned State TB Officer (STO), IRL and NRL using an IRL OSE Checklist while OSE checklist for NRL Laboratory Personnel to IRL is prepared using a NRL OSE Checklist.

Resources

RNTCP Laboratory Network Guidelines.

Assessment

Documentation of any significant problems (technical, operational, others) and feedback for corrective action during On-site Evaluation (OSE) is necessary to formulate plans with the District TB Officer (DTO), Senior TB Laboratory Supervisor (STLS), Medical Officer (MO), Intermediate Reference Laboratory (IRL), National Reference Laboratory (NRL) and Laboratory Technician (LT) to improve the quality of smear microscopy.

Checklists for OSE

• OSE is done through comprehensive checklists.
• The checklist must be used by NRL, IRL and District TB Center (DTC) STLS and a shorter version by DTO/ Medical Officer TB Center (MOTC) during each OSE visit to document observations and corrective action.
• The checklists contain open, non-leading questions and recommended observations along with objective criteria for acceptable practices so that the supervisor can assess how well the technician understands proper procedures.

Feedback

Feedback is the process of communicating results of External Quality Assurance (EQA) to the original laboratory, including suggestions for possible causes of errors and remedies.

TB Unit On-site Evaluation (TU-OSE) Checklist and Feedback

The observations in TU-OSE Checklist, including observation of five positive and negative slides must be documented (Figure 1). Corrective actions must be discussed by the STLS with LT and checklist should be signed by MO of DMC.

1: Write smear and grade
2: Tick appropriate column
3: Tick if good; write ‘U’ if under-decolourized, ‘O’ if over-decolourized
4: Tick if good; write ‘B’ if too big, ‘S’ if too small
5: Tick if good; write ‘K’ if too thick, ‘N’ if too thin
6: Tick appropriate column

Figure 1: Review of five positive and five negative slides by STLS and instructions to fill observations in TU-OSE Checklist; Source: RNTCP Laboratory Network Guidelines

Action Required based on TU-OSE Checklist

The STLS should:

• Enter the summary of ‘action required’ in the Supervision Register before leaving the DMC
• Submit the summary report of DMCs under him to DTO on a monthly basis
• Maintain separate files for checklists including summary reports of each DMC in respective TU and submitted for review by higher level supervisors including on-site evaluation visits by the IRL to the district.

The DTO should:

• Give monthly feedback to the MO in-charge of the respective DMC
• Send summary of results of checklists for each DMC in the district once every quarter for analysis by the respective CMO/ DMO and IRL.

Checklist IRL OSE and NRL OSE and Feedback

• On-site quarterly evaluation report of DTCs visited by IRL team should be given to concerned State TB Officer (STO), IRL and NRL using an IRL OSE Checklist.
• OSE Checklist for NRL Laboratory Personnel to IRL is prepared using a NRL OSE Checklist.
• The IRLs will submit a consolidated quarterly report to the concerned STO and NRL. In turn, the NRL will submit a consolidated quarterly report to the Central TB Division.
• A comprehensive list of all operational elements to be observed will help to ensure consistency in laboratory evaluations and provide immediate feedback to the technicians to facilitate rapid corrective action, as well as serve as documentation of the visit and record of current conditions and actions needed.
• All potential sources of error should be investigated, including quality of stains and staining procedure, quality of microscopes, and administrative procedures that may contribute to recording errors and corrective actions provided (Figure 2).
• All problems contributing to errors must be resolved.
• Possible causes of errors, and suggested evaluation steps are provided in Annexure K.
• Remedial training must be provided for technicians unable to properly identify AFB in smears.

Figure 2: Documentation of corrective actions in IRL-OSE Checklist )RNTCP Laboratory Network Guidelines)

Action Required for IRL/NRL-OSE Checklist

A state level consolidated summary will be prepared by the respective IRL every quarter from the district summaries for submission to STO and NRL. The DTO/ CMO of the district is to submit an action taken report on the team’s recommendations to the STO within a month of the IRL visit.

Resources

RNTCP Laboratory Network Guidelines.

Assessment