Laboratory Identification of Tuberculosis PDF

Laboratory identification of tuberculosis and Updates on direct sputum smear microscopy  Laboratory identification of tuberculosis Tuberculosis (TB) is a contagious bacterial infection that primarily affects the lungs but can also impact other parts of the body. Accurate and...

Laboratory identification of tuberculosis and Updates on direct sputum smear microscopy  Laboratory identification of tuberculosis Tuberculosis (TB) is a contagious bacterial infection that primarily affects the lungs but can also impact other parts of the body. Accurate and timely diagnosis is crucial for effective treatment and prevention of TB transmission. Laboratory identification plays a vital role in the diagnostic process, providing evidence of the presence of Mycobacterium tuberculosis (Mtb) in clinical samples. The laboratory identification of TB relies on a combination of techniques, including direct sputum smear microscopy (DSSM), culture-based methods, and molecular assays.  Mycobacterium Mycobacteria are aerobic, non Mycobacterium tuberculosis is Liquid media are Dubos’, motile, nonencapsulated and non weakly gram-positive, strongly Middlebrook’s, Proskauer and sporing. Growth is generally slow acid-fast, aerobic bacilli Beck’s, Sula’s and Sauton’s Mycobacteria are, known as acid- media are the more common fast bacilli (AFB). Acid fastness L–Jmedia, M. tuberculosis has been ascribed variously to the forms dry, rough, raised, They are weakly catalase presence in the bacillus of irregular colonies with a positive, neutral-red positive, mycolic acid or to a wrinkled surface. amidase positive, nitrate semipermeable membrane reduction test positive, niacin around the cell test negative, and arylsulfatase negative Epidemiology and tuberculosis Pathogenesis and immunity: The source of infection is usually an open case of pulmonary tuberculosis. The initial infection with M. tuberculosis is referred to as a primary infection. Subsequent disease in a previously sensitized person, known as post primary (secondary or reinfection) Worldwide, TB is the 13th leading tuberculosis cause of death and the second leading infectious killer after COVID-19 (above HIV/AIDS). Diseases: M. tuberculosis causes total of 1.5 million people died primarily pulmonary tuberculosis from TB in 2020 (including 214 000 people with HIV). In 2020, an estimated 10 million Diagnosis: Bacteriological diagnosis people fell ill with tuberculosis of tuberculosis can be established by (TB) worldwide. 5.6 million men direct microscopy, culture examination 3.3 million women and or by animal inoculation test 1.1 million children Epidemiology and tuberculosis There is 30 high TB burden countries accounted for 86% of new TB cases Eight countries account for two thirds of the total PHILIPPINES TUBERCULOSIS ROADMAP population OVERVIEW, FISCAL YEAR India 2021 China, Estimated to have a Indonesia 599,000 active tb case Philippines, Ranked as seventh in the Pakistan, MDR TB burden countries Nigeria, Bangladesh and South Africa Epidemiology and tuberculosis In 2023, tuberculosis (TB) continues to be a significant global health concern. Worldwide, there has been an increase in TB cases, largely attributed to the disruptions caused by the COVID-19 pandemic. The World Health Organization's Global Tuberculosis Report highlights that TB cases globally rose to around 10.6 million in 2021, a stark contrast to earlier progress towards eradicating the disease. In the Philippines, TB remains a pressing issue, being one of the eight countries that account for two-thirds of global TB cases. In 2022, the Department of Health (DOH) reported over 372,000 cases, a figure that underscores the challenge the country faces in combating the disease. Case finding Case finding is the identification of presumptive TB, either by clinical signs and symptoms or chest X-ray, followed by the diagnosis of active TB disease through bacteriological testing or clinical diagnosis. Presumptive TB can be identified through systematic screening in health facilities, or among targeted populations in congregate settings, the community or workplaces by using either symptom-based screening, chest X-ray or both. The bacteriological test recommended is a rapid diagnostic test (e.g. Xpert MTB/RIF).  Policies 1.Systematic screening for TB 1.Symptom screening using any of shall be implemented in all the four cardinal signs and symptoms (at least two weeks of cough, health facilities. unexplained fever, unexplained weight loss and night sweats) shall be 1.Screening by chest X- 2.Systematic screening for the primary screening tool for ray shall be recommended active TB – refers to the systematic screening in health annually among all health systematic identification of facilities among all consults including presumptive TB in a for immunization, maternal health and facility consults. child health. Accompanying persons predetermined target group, will also be screened by asking for TB using examinations or other signs and symptoms. procedures that can be applied rapidly.  Policies 1.Active case finding shall be implemented in 1.All health facilities shall 1.All people living with congregate settings, screen its workers for TB targeted communities and HIV (PLHIV) shall be screened for the TB co- annually using both workplaces using chest X- symptom and chest X-ray ray as the primary infection. screening. screening tool and Xpert as the diagnostic test.  Systemic screening in health facilities (intensified case finding) Systematic screening in facilities shall be done for all clients visiting the facility regardless of reason for consult. The following steps are involved in screening for pulmonary TB (PTB) in adults ≥ 15 years old Ask all patients consulting the health facility, if they have the following cardinal signs and symptoms that are lasting for ≥2 weeks: Cough Unexplained Fever Weight loss Night Sweats  remember If any of the above signs/symptoms are present for at least two weeks, identify as a presumptive TB. For those who do not have any of the cardinal signs/ symptoms above or experienced it for less than two weeks, offer chest X-ray screening if one has not been conducted in the past year. The National TB Prevalence Survey in 2016 showed that “screening for TB cases using symptoms alone would have missed one-third to two-thirds of bacteriologically confirmed pulmonary TB cases.” !!! Roles and Functions of Medical Technologist Involved in NTP  Case finding is the identification and diagnosis of TB cases among individuals with signs and symptoms presumptive of tuberculosis. The current approach to case finding includes passive and intensified case finding. The available tests utilized by the program for diagnosing TB are direct sputum smear microscopy, TB culture and drug susceptibility test, tuberculin skin test and rapid molecular diagnostic tests. Direct sputum smear microscopy (DSSM) is fundamental to the DSSM serves as one of the 1. It provides a definitive bases for categorizing TB cases detection of infectious cases and diagnosis of active TB; according to standard case is recommended for case finding 2. The procedure is simple; definition. This is also used to: among adults and children who 3. It is economical; and, a) monitor progress of patients can expectorate. It is the primary 4. A microscopy center could be with TB while they are on diagnostic method adopted by put up even in remote areas. anti– TB treatment; the NTP among such individuals because: b) b) confirm cure at the end of treatment.  CLASSIFICATION OF TB DISEASE 1. Classification based on bacteriological status a. Bacteriologically-confirmed – A TB patient from whom a biological specimen is positive by smear 2. Classification based on anatomical site microscopy, culture or rapid diagnostic tests (such as Xpert MTB/RIF). 1.Pulmonary TB (PTB) – Refers to a case of tuberculosis involving the lung parenchyma. A patient with both pulmonary and extra-pulmonary TB should be b. Clinically-diagnosed – A PTB patient who does classified as a case of pulmonary TB. not fulfill the criteria for bacteriological confirmation but has been diagnosed with active TB by a clinician or 2.Extra-pulmonary TB (EPTB) – Refers to a case of tuberculosis involving other medical practitioner who has decided to give the organs other than the lungs (e.g., larynx, pleura, lymph nodes, abdomen, patient a full course of TB treatment. This definition genito- urinary tract, skin, joints and bones, meninges). Histologically-diagnosed includes cases diagnosed on the basis of CXR EPTB through biopsy of appropriate sites will be considered clinically-diagnosed abnormalities or suggestive histology, and extra- TB. Laryngeal TB, though likely sputum smear-positive, is considered an extra- pulmonary cases without laboratory confirmation. pulmonary case in the absence of lung infiltrates on CXR. POLICIES Policies for dssm Smear microscopy (whether brightfield or Available rapid diagnostic test (e.g., Xpert MTB/RIF) shall be fluorescence microscopy) or loop mediated used for TB diagnosis among isothermal amplification (TB LAMP) shall be presumptive DR-TB, PLHIV with the alternative diagnostic test if Xpert is not signs and symptoms of TB, accessible. Unavailability of Xpert MTB/RIF smear-negative adults with CXR test shall not be a deterrent to diagnose TB findings suggestive of TB smear-negative children and Two sputum specimens of disease bacteriologically. EPTB. good quality shall be collected, either as frontloading (i.e., spot-spot one-hour apart) or spot- early morning specimens, based on the patient’s preference. The two specimens should be TB LAMP may be collected at most within 3 utilized to process days. large sample loads especially in ACF activities, but not for children, PLHIV and MDR-TB risk groups Policies for dssm Health facilities with TB services, whether All PLHIV shall be public or private, are encouraged to establish screened for TB co- their own in-house TB diagnostic laboratory infection. such as Xpert MTB/RIF, SM and TB LAMP. In cases where it is not possible, access to an officially NTP-linked TB diagnostic laboratory would be acceptable. All laboratories providing DSSM services or other TB diagnostic tests, whether public or private, shall participate in the External Trained health workers shall do the Quality Assessment (EQA) testing and reading of TST. An system of the NTP. induration of at least 10 mm regardless of bacille Calmette- Guerin (BCG) vaccination status or 5 mm in immunocompromised children (e.g. severely malnourished) is considered a positive TST reaction.  MAIN FEATURES Specimen collection Motivate the presumptive TB to 2. undergo DSSM. Explain the importance of the procedure and that 1. For Xpert MTB/RIF, prepare a of submitting two (2) sputum sputum cup or 50ml conical Prepare the sputum cups and tube and Form 2a. NTP specimens. The only contraindication the Form 2a. NTP Laboratory Request Form. to collecting sputum for DSSM is Laboratory Request Form. Label the body of the sputum massive hemoptysis which is Label the body of the sputum cup/conical tube, indicating cup (i.e., not the lid), patient’s complete name and expectoration of large volumes of indicating patient’s complete indicating specimen for Xpert. blood (200-600 ml in 24 hours) from name, and order of specimen the respiratory tract. collection (i.e., 1st, and 2nd). Blood streaked sputum can still be 4. examined. 3. Observe proper precautions against infection during the demonstration. Stay behind the Demonstrate how to produce quality sputum. patient. Collect specimen in a well- Mucus from the nose and throat, and saliva from ventilated designated sputum the mouth are NOT good specimens. Advise the collection area, or outside the patient to: DOTS facility.  MAIN FEATURES Specimen collection 6. Motivate the presumptive TB to undergo DSSM. Explain the 5. Check quantity and quality of importance of the procedure and that sputum. Wipe off the external Collect the first specimen (i.e., surface of the sputum cup or of submitting two (2) sputum spot) at the time of the first conical tube if needed and specimens. The only contraindication consultation. Collect the second wash your hand thoroughly spot specimen after at least an to collecting sputum for DSSM is with soap and water. hour, or the following morning. If massive hemoptysis which is the second sputum specimen is not submitted within three days expectoration of large volumes of from the first specimen, a new set blood (200-600 ml in 24 hours) from of two (2) sputum specimens should be collected unless the the respiratory tract. first specimen already tests positive for AFB. 8. Blood streaked sputum can still be 7. 4.Inform the patient when to examined. return for follow-up consultation regarding the Seal the sputum cup or conical tube, pack it results. TYPES OF SAMPLE securely, and transport it to a microscopy center or 1. Spot-spot Xpert MTB/RIF site together with the completely 2. Early morning – Spot filled up Form 2a. NTP Laboratory Request 3. Early morning – Early morning Form. a. Clean mouth by thoroughly rinsing with water. Food particles or other solid particulates may inhibit the test for Xpert MTB/RIF. b. Breathe deeply, hold breath for a second or two, and then exhale slowly. Repeat the entire sequence two (2) more times. c. Cough strongly after inhaling deeply for the third time and try to bring up sputum from deep within the lungs. GOOD QUALITY d. Expectorate the sputum in the sputum cup or conical tube. SPUTUM e. Collect at least 1 teaspoonful (5-10ml) for DSSM. For Xpert MTB/RIF, sputum sample should not be less than one (1) ml. f. Examine the specimen to see that it is not just saliva. Repeat the process if necessary. Gargle with lukewarm Prior to collection: distilled water only. Do not gargle with mouthwash. Brushing your teeth is not recommendable. Do not eat. Do not drink (i.e., coffee, alcohol, tea etc.) VISUAL APPEARN CE OF SPUTUM PROCEDUR E FOR DSSM DSSM may be performed using either conventional Ziehl- 1.Record the patient information in the Form 3. NTP Laboratory Register (Microscopy and GX). Neelsen microscopy or 2.Smear, fix, and stain each slide. fluorescence microscopy (FM). 3.Read each slide and interpret the result. Fluorescence microscopy using 4. Interpret the results of the two light-emitting diodes (LED) as specimens and write the final laboratory diagnosis in the lower the microscope light source is portion of Form 2a. NTP also known as LED-FM. Laboratory Request Form for Fluorescence microscopy has DSSM and on the Remarks column of Form 3. NTP Laboratory increased sensitivity and can Register (Microscopy and Xpert be five (5) times faster. MTB/RIF). 5. Send the request form with its corresponding results back to requesting unit within three (3) working days.  AFB SMEAR 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. 1. Using the uneven end, select and pick purulent portions of the sputum Use new, clean, unscratched Frosted glass slides and specimen and transfer onto a new, label the slide with the laboratory serial number. clean, labelled, glass slide. 2. Using the wooden stick, spread the Prepare the smear in the centre of the slide covering sputum evenly, in a continuous 3 cm X 2 cm rotational movement, to cover two- thirds of the central portion of the slide. Smear preparation should be The smear is prepared by using either a wooden stick or done near a flame. This is required a disposable loop. as approximately 6 inches around the flame is considered as a sterile zone which coagulates the aerosols raised during smear preparation. ZIEHL NEELSEN AFB STAIN - It is used to stain bacteria that have high lipid content in their cell walls. Principle: The primary stain binds to mycolic RULE AFTER AFB acid in the cell walls of the mycobacteria and is retained after STAINING decolorozation with acid alcohol. ALL BACTERIA ARE NON- Acid-fast bacilli (AFB) retain the primary stain and are colored red ACID FAST EXCEPT: (carbol fuschin) Mycobacteria, non-AFB blue or green (methylene Nocardia, Rhodococcus, blue or malachite green Tsukamurella, Gordonia and counterstains) color. (resistant to decolorization) Legionella micdadei.  AFB STAINING METHOD A. Prepare and fix the specimen smear prior to staining. B. Flood the slide with aqueous carbol fuchsin solution. Using a low flame, steam it for 10 minutes. Make sure that the preparation does not dry up by placing a strip of blotting paper or filter paper onto the slide and adding more carbol fuchsin from time to time. C. Wash off the excess stain with water. D. Decolorize the smear with acid alcohol by adding the alcohol in small amounts (drop by drop) for 30 seconds until no more stain comes off with the alcohol. E. Counterstain with Methylene blue for 60 seconds. F. Wash off the excess stain with tap water. G. Air or blot dry and examine under OIO (1000x total magnification).  Air drying and heat fixing sputum smear The procedure for air-drying and heat-fixing the slide is as follows: 1. A smear prepared on a clean glass slide from mucopurulent portion of the specimen is air dried for 15-30 minutes on a rack Step 2 2. 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 for 3-4 seconds each time. 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. Do’s and Don’ts for a Good Quality Smear 1. Do ensure that the smear size is 3 cm by 2 cm 2. Do ensure there are no fingerprints on the prepared smear 3. The smear should not be very thick, but it should be thin enough to visualize a newsprint  Ways to facilitate AFB Stain 1. 2 3 4 Heating/Steaming By Increasing the concentration of Process - to temporarily dye and phenol. Prolonged contact Addition of a. Ziehl-Neelsen = 3 grams of the specimen wetting agent remove mycolic acid Carbolfuchsin + 5% phenol while the smear is b. Kinyoun's = 4 grams with the primary (Tergitol). flooded with stain. Carbolfuchsin + 9% phenol stain.  MICROSCOPY NUMBER ACCESSION LAAB DATE PATRICK MINA A. Read the whole slide from left AFB 1 to right. This is equivalent to 150 fields. B. Go down 1 field and read it from right to left. This is equivalent to another 150 fields for a total of 300 fields. C. Record your results. Positive = at least one sputum smear is positive for AFB (+n, 1+, 2+, 3+) Negative = both sputum smears are negative for AFB. Causes of False-positive Acid-fast Smear: 1. Changes in the cell wall. 2. Insufficient decolorization. 3. Laboratory contamination. 4. Delayed processing and overgrowth of other bacteria. Causes of False-negative Acid-fast Smear: 1. Overzealous decontamination. 2. Loss from concentration techniques. 3. Organism obscured by a very thick smear. 4. Over decolorization. 5. Poor counterstaining. 6. Lack of observer proficiency in reading sputum smears.  Recent updates Have primarily focused on improving accuracy, speed, and accessibility in the diagnosis of tuberculosis (TB). 1. Fluorochrome Staining (Auramine-O) a. Higher Sensitivity: Compared to the traditional Ziehl-Neelsen stain, Auramine-O staining, used with fluorescence microscopy, has shown greater sensitivity for detecting TB bacteria. b. Faster Reading: Fluorochrome-stained slides allow for rapid scanning under lower magnification, speeding up the diagnosis process.  Recent updates LED Fluorescence Microscopy a. Energy Efficiency: LED fluorescence microscopy is more energy-efficient than traditional fluorescent lamps, making it suitable for resource-limited settings. b. Improved Detection Rates: Studies have shown that LED microscopy increases TB detection rates, particularly in low-resource settings where conventional microscopy may not perform as well. c. Longevity and Cost-Effectiveness: LEDs last longer and are cheaper to maintain, reducing operational costs in the laboratory. Recent updates Digital Microscopy - Automation: Some labs are adopting digital microscopy systems that automate smear scanning, reducing human error and increasing diagnostic throughput. - Image Archiving and Remote Diagnosis: Digital systems allow for image archiving and remote review, enabling external quality control and consultations, even from distant experts.  ritm RITM Proficiency Testing for AFB in the Philippines is a critical component Key Features of RITM Proficiency Testing of the National Tuberculosis Control Program (NTCP) aimed at for AFB in the Philippines: maintaining and improving the accuracy and reliability of tuberculosis (TB) diagnosis using Direct Sputum 1. Evaluation of Laboratory Competency: Smear Microscopy (DSSM). 2. Quality Assurance Process: 3. Accreditation and Certification: Conducted by the Research Institute for Tropical Medicine (RITM) this 4. External Quality Assessment (EQA): testing ensures that laboratories 5. Improving TB Control: performing AFB smear microscopy meet international standards and provide consistent results.  Positive AFB – Mycobacterium tuberculosis ATCC 25177 QUALITY Positive- Partially AFB – CONTROL Nocardia asteroides ATCC SLIDES 19247 Negative – Non AFB – Escherichia coli ATCC 25922  GENEXPERT is a molecular diagnostic test 1.PCR Amplification: primarily used for the detection of Mycobacterium tuberculosis (MTB) and its resistance to - GeneXpert uses real-time 1.Cartridge based system rifampicin (RIF), a key TB drug. PCR to amplify specific DNA 2.Uses Fluorescent The test is based on real-time sequences of Mycobacterium Probes polymerase chain reaction tuberculosis. (PCR) technology and is widely 3.Multiplex testing - The system targets the rpoB used in TB diagnosis due to its gene in the M. tuberculosis speed, accuracy, and ability to complex (MTBC), which is detect drug resistance. crucial for rifampicin resistance.  GENEXPERT Result Interpretation: - The GeneXpert system automatically analyzes the results and provides an easy-to-interpret output within 2 hours. - Results typically indicate: -1. MTB detected/not detected 2. RIF resistance detected/not detected 3. Indeterminate result (if there is an issue with the sample or assay) Source: