Organization of detection and diagnostics of tuberculosis.

1. PHEE Kyiv Medical
University
LECTURE №2.
Organization of detection and diagnostics of
tuberculosis.
Lector: Ivashchenko Oleksandr Andriiovych, assistant of Infectious diseases, pulmonology and phthisiology department.

2. Mission, vision and values
MISSION
In relentless pursuit of excellence, to teach, to search, to heal and to
serve humanity
VISION
To transform health care for the benefit of the people and communities
by becoming a national leader in educating health care professionals
VALUES
Excellence, innovation, commitment, integrity, respect, accountability

3. Detection of tuberculosis is an application of the
active (a fluorography, smear microscopy, a
tuberculinodiagnostics) or passive methods
(collecting complaints and the anamnesis, a
physical, X-ray inspection and a double
microscopy of dab of a sputum) for the purpose of
establishment of the preliminary diagnosis of
tuberculosis which is necessary for sorting or
selection of persons with suspicion of tuberculosis,
development and implementation of the plan of
inspection. Identification of TB patients is a
prerogative of any doctor of the common medical
network.
• Detection of tuberculosis-1

4. Detection of tuberculosis can be:
• Early identification – is diagnosis of the tubercular intoxication
arising due to primary infection.
• Well-timed is an identification of forms of tuberculosis which is
oligosymptomatic, without phase of disintegration and excretion of
MTB, with localization of process within 1-3 segments at which
treatment 100% of convalescence with minor residual changes reach.
Well-timed identification most often meets at primary tuberculosis,
focal, infiltrative, disseminated pulmonary tuberculosis, exudative
pleurisy.
• Untimely – identification of primary or secondary forms of
tuberculosis with existence of a cavity of disintegration and (or)
bacterial excretion. Effectiveness of treatment - 65-85%. After an
adhesion there are expressed residual changes in a type of
widespread sites of a fibrosis, bronchiectasis, and tuberculomas.
• Late (the started tuberculosis) - identification of the heavy and
started tuberculosis forms (fibrous-cavernous, chronic disseminated,
cirrhotic tuberculosis, a pleura empyema). Effectiveness of treatment
is low - 12-25%. The constant or periodic bacterial excretion is
observed that is epidemiologically dangerous.
• Detection of tuberculosis-2

5. Increasing sanitary literacy of the population.
Everyone has to know:
• Tuberculosis is an infectious disease;
• Main symptoms of tuberculosis;
• Ways of detection from tuberculosis;
• Features of a current and treatment of tuberculosis.
Improving skills of medical personnel. Doctors, paramedics and
nurses have to:
• To know incidence and prevalence of tuberculosis in the country;
• To know features of tuberculosis of various forms and
localizations;
• To be vigilant and alerted on tuberculosis;
• To be able to reveal the TB patient of various forms and
localizations.
Developing possibilities of social support of patients (especially
aged, disabled people, refugees, the homeless).
• Early identification

6. • Medical history and physical examination
• Tuberculin Skin Test (TST) or Interferon-Gamma Release
Assays (IGRAs)
• Chest X-ray
• Sputum microscopy
• Bacteriological culture
• Nucleic Acid Amplification Tests (NAATs)
• Other diagnostic tools
• Diagnosis of tuberculosis

7. During the medical history, the healthcare provider typically asks questions related to the
following aspects:
• Symptoms: the patient is asked about any symptoms they may be experiencing, such as
persistent cough (often lasting more than two weeks), sputum production, chest pain,
fatigue, night sweats, unexplained weight loss, fever, or hemoptysis (coughing up blood).
These symptoms raise suspicion for active TB disease.
• Duration of symptoms: the duration of symptoms is important in distinguishing acute
respiratory infections from chronic TB. Symptoms lasting for more than two weeks are
more suggestive of TB.
• Risk factors: the healthcare provider assesses the patient's risk factors for TB, which
include:
1. Close contact with a known TB case: Living or working closely with someone who
has active TB disease.
2. Travel or residence in areas with high TB prevalence: Certain regions or countries
where TB is more common.
3. Immunocompromised status: HIV infection, organ transplantation, certain medical
conditions (e.g., diabetes, chronic kidney disease), or immunosuppressive
medications.
4. Healthcare workers: Occupational exposure to TB patients.
5. Homelessness or incarceration: Conditions associated with higher TB transmission
rates.
6. Substance abuse: Intravenous drug use, which can increase the risk of TB infection.
• Past medical history: the healthcare provider asks about any previous TB infection or
treatment, as well as other medical conditions that may affect the immune system or
increase the risk of TB.
• Diagnosis of tuberculosis (Medical history)

8. The physical examination focuses on identifying signs and
findings that may suggest active TB disease. Key aspects of
the examination include:
• Respiratory system: the healthcare provider assesses the
patient's respiratory function, listening for abnormal lung
sounds (such as crackles or decreased breath sounds) that
may indicate lung involvement. They also evaluate for signs
of respiratory distress or abnormal respiratory patterns.
• Lymph nodes: the provider palpates the lymph nodes,
especially those in the neck, supraclavicular region, and
axilla, to check for enlargement, which can be a sign of TB
dissemination.
• Other systemic examination: depending on the patient's
symptoms and clinical presentation, the healthcare provider
may perform a thorough examination of other body systems
to identify any additional signs of TB involvement, such as
hepatomegaly (enlarged liver), splenomegaly (enlarged
spleen), or joint abnormalities.
• Diagnosis of tuberculosis (Physical examination)

9. The Tuberculin Skin Test (TST), also known as the
Mantoux test, is a widely used diagnostic test for
tuberculosis (TB) infection. It is based on the principle
of delayed hypersensitivity reaction to antigens derived
from Mycobacterium tuberculosis (M. tuberculosis).
• Administration:
The TST involves the intradermal injection of a small
amount (0.1 mL or 2 TU PPD-L) of purified protein
derivative (PPD) tuberculin into the inner surface of
the forearm. The injection is usually performed using a
small needle, creating a wheal or blister-like raised
area on the skin.
• Reading and timing:
The test is read and evaluated 48 to 72 hours after
administration. This timing is crucial, as the reaction
takes time to develop.
• Diagnosis of tuberculosis (TST)-1

10. Interpretation of results.
The TST reaction is measured by the size of the induration (raised, hardened area) at the injection site. Induration is the key
parameter used to determine the test result, rather than erythema (redness) or other skin reactions. The induration is measured
in millimeters using a ruler.
• Diagnosis of tuberculosis (TST)-2

11. Interferon-Gamma Release Assays (IGRAs) are whole-blood
tests that can aid in diagnosing Mycobacterium tuberculosis
infection. They do not help differentiate latent tuberculosis
infection (LTBI) from tuberculosis disease. Two IGRAs that
have
been approved by the U.S. Food and Drug Administration
(FDA):
• QuantiFERON® – TB Gold In-Tube test (QFT–GIT);
• SPOT® TB test (T–Spot)
IGRAs measure a person’s immune reactivity to M.
tuberculosis. White blood cells from most persons that have
been infected with M. tuberculosis will release interferon-
gamma (IFN-g) when mixed with antigens (substances that can
produce an immune response) derived from M. tuberculosis. To
conduct the tests, fresh blood samples are mixed with antigens
and controls. The antigens, testing methods, and interpretation
criteria for IGRAs differ.
What are the advantages of IGRAs?
• Requires a single patient visit to conduct the test.
• Results can be available within 24 hours.
• Does not boost responses measured by subsequent tests.
• Prior BCG (bacille Calmette-Guérin) vaccination does
not cause a false-positiveIGRA test result.
• Diagnosis of tuberculosis (IGRAs)

12. Chest X-ray is a commonly used imaging technique to evaluate the lungs and is an important tool in the
diagnosis of tuberculosis (TB). Here are more details about chest X-ray findings in tuberculosis:
• Purpose:
A chest X-ray is performed to assess the lungs for abnormalities that may suggest active TB disease or
complications of TB, such as lung cavities, infiltrates, or lymph node enlargement. It helps in confirming a
TB diagnosis, evaluating the extent of disease, and monitoring treatment response.
• Imaging findings:
The chest X-ray findings in tuberculosis can vary depending on the stage and severity of the disease.
Common findings include:
• Infiltrates: in active pulmonary TB, the X-ray may show areas of consolidation or infiltrates, which
appear as hazy opacities or white patches in the lung fields. These infiltrates indicate the presence
of active inflammation and infection in the lung tissue.
• Cavities: in some cases, TB infection can cause the formation of cavities within the lung tissue.
Cavities appear as air-filled spaces or cavities with irregular or thickened walls on the X-ray.
Cavities are more commonly seen in advanced or chronic TB.
• Ghon focus and Ghon complex: these terms are used to describe specific patterns seen in the lungs
during primary TB infection. A Ghon focus refers to a small area of infection typically seen in the
lower part of the lungs. A Ghon complex is a combination of a Ghon focus and associated lymph
node enlargement (hilar or mediastinal lymphadenopathy).
• Pleural effusion: TB infection can sometimes lead to the accumulation of fluid in the pleural space
surrounding the lungs, resulting in a pleural effusion. On the X-ray, this appears as a blunting or
opacity of the costophrenic angle, which is the sharp angle formed by the diaphragm and the
ribcage.
• Lymphadenopathy: TB can cause enlargement of the lymph nodes in the chest, particularly the
hilar and mediastinal lymph nodes. Enlarged lymph nodes may appear as round opacities or masses
on the X-ray.
• Diagnosis of tuberculosis (Chest X-ray)
Post-primary TB
(secondary TB or reactivation TB)

13. • Chest X-ray-1

14. • Chest X-ray-2
This patient has a lung cavity which was initially thought to be a cancer but which disappeared after
a course of antibiotics

15. • Chest X-ray-3
The right middle lung zone shows a dense nodular opacity
The right middle lung zone shows a dense nodular
opacity measuring about 10 mm
GHON FOCUS

16. Bilateral Lymphadenopathy
• Chest X-ray-4

17. • Chest X-ray-5
Healed post-primary TB
• Following an immune response to post-primary infection, the
affected area often becomes scarred (fibrotic) and calcified.
• The combined fibrosis and calcification can be described as “fibro-
calcific change”.

18. Sputum microscopy is a commonly used diagnostic test for
tuberculosis (TB) that involves examining sputum samples under a
microscope to detect the presence of acid-fast bacilli (AFB), primarily
Mycobacterium tuberculosis (M. tuberculosis).
• Purpose:
Sputum microscopy is performed to identify the presence of AFB in
the sputum, indicating the possible presence of active TB disease. It
is a rapid and cost-effective method for initial screening and can help
guide further diagnostic investigations.
• Sample collection:
Sputum samples are collected from individuals suspected of having
TB. The patient is instructed to provide an early morning sputum
sample, as it tends to be more representative of the respiratory tract
and has a higher yield of AFB. The individual is asked to cough
deeply and expectorate sputum directly into a sterile container.
• Sample preparation:
Once the sputum sample is collected, it undergoes preparation before
microscopy. The sputum is mixed with a decontamination solution,
usually sodium hypochlorite or N-acetyl-L-cysteine (NALC)-sodium
hydroxide, to break down any mucous or debris and kill other
microorganisms present in the sputum. This step helps concentrate
the AFB for better visualization.
• Diagnosis of tuberculosis (Sputum microscopy)-1

19. Acid-Fast staining:
After the sample preparation, the sputum is placed on a glass slide and subjected to
an acid-fast staining technique, typically the Ziehl-Neelsen or Kinyoun method.
The staining procedure involves the following steps:
1. The sputum sample is heat-fixed onto the slide.
2. It is then flooded with a primary stain called carbol fuchsin, which stains the
AFB.
3. The slide is heated to allow the stain to penetrate the mycobacterial cell wall.
4. After rinsing, the slide is treated with acid-alcohol to remove the stain from non-
acid-fast organisms.
5. Finally, a counterstain, such as methylene blue, is applied to stain the remaining
bacteria.
Microscopic examination:
The stained slide is observed under a microscope, typically using an oil-immersion
lens, at a magnification of 1000x. The examiner looks for the presence of red-stained
AFB against a blue background. AFB appear as slender, rod-shaped organisms with a
characteristic beaded or "cording" appearance. The number of AFB per field is
recorded.
Interpretation of results:
The results of sputum microscopy are reported as the presence or absence of AFB and
the quantity observed. The interpretation of the results depends on the number of
AFB seen in the microscopic field. The general categories used for reporting are:
a. Positive (+): AFB are detected and quantified, typically reported as 1+, 2+, 3+, or 4+
based on the number of AFB observed per field. Higher grades indicate a higher
bacterial load.
b. Negative (-): No AFB are seen in the sputum sample after examining at least 100
fields. However, a negative result does not definitively rule out TB, as the test has
limitations, including variable sensitivity.
• Diagnosis of tuberculosis (Sputum microscopy)-2

20. Bacteriological culture is a diagnostic method for tuberculosis (TB) that involves the isolation
and growth of Mycobacterium tuberculosis (M. tuberculosis) in a laboratory setting. It allows
for the definitive identification of the bacteria and the determination of its drug susceptibility.
• Purpose:
Bacteriological culture is used to confirm the presence of M. tuberculosis in clinical samples,
such as sputum, bronchoalveolar lavage, or other bodily fluids, and to assess its drug
susceptibility. It is considered the most accurate method for TB diagnosis and plays a crucial
role in guiding treatment decisions.
• Sample collection:
Clinical samples are collected from individuals suspected of having TB. The most common
sample is sputum, but other samples may be collected depending on the site of infection, such
as cerebrospinal fluid, pleural fluid, or lymph node aspirate. Proper collection techniques and
sample handling procedures are followed to minimize contamination and ensure sample
viability.
• Sample processing:
The collected sample undergoes various processing steps to prepare it for culture:
1. Decontamination: the sample is decontaminated to eliminate potential contaminants
and non-mycobacterial organisms. Common decontamination methods include N-
acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and sodium hydroxide (NaOH)
procedures.
2. Concentration: after decontamination, the sample is centrifuged to concentrate the
mycobacteria present in the sample. The sediment is then resuspended in a small
volume of liquid medium.
3. Inoculation: the concentrated sediment is inoculated onto appropriate culture media
specifically designed to support the growth of M. tuberculosis. The most commonly
used culture media for TB include solid media such as Lowenstein-Jensen (LJ) or
Middlebrook 7H11 agar, as well as liquid media like Middlebrook 7H9 broth or
mycobacteria growth indicator tube (MGIT) system.
• Diagnosis of tuberculosis (Bacteriological culture)-1
Middlebrook 7H11 Agar
Lowenstein-Jensen

21. • Incubation and growth:
The inoculated culture media are placed in a controlled incubator at a suitable temperature (typically 37°C) to provide optimal conditions
for the growth of M. tuberculosis. The cultures are monitored regularly for growth and observed for visible colonies or turbidity in the liquid
media. The incubation period can range from a few days to several weeks, depending on the growth rate of the bacteria.
• Identification:
Once growth is observed, further tests are conducted to confirm the identity of the bacteria as M. tuberculosis. This may involve specific
staining techniques, such as acid-fast staining, to visualize the acid-fast bacilli and confirm their mycobacterial nature. Molecular
techniques, such as polymerase chain reaction (PCR), may also be used to detect specific genetic markers unique to M. tuberculosis.
• Drug susceptibility testing:
In addition to identification, culture allows for drug susceptibility testing (DST) to determine the sensitivity or resistance of the isolated M.
tuberculosis strains to various anti-TB drugs. This information is critical for selecting the most effective treatment regimen for individual
patients.
• Diagnosis of tuberculosis (Bacteriological culture)-2

22. Nucleic Acid Amplification Tests (NAATs) are advanced molecular diagnostic techniques used for the detection of
Mycobacterium tuberculosis (M. tuberculosis) and the diagnosis of tuberculosis (TB). NAATs offer high sensitivity and
specificity and provide rapid results compared to traditional culture-based methods. Here are more details about
Nucleic Acid Amplification Tests for TB:
• Principle:
NAATs are based on the principle of amplifying and detecting specific nucleic acid sequences of M. tuberculosis
present in clinical samples. The most commonly targeted nucleic acid targets are the genes that encode for the 16S
ribosomal RNA or the mycobacterial-specific insertion sequence IS6110. These targets are highly specific to M.
tuberculosis and allow for accurate identification.
• Sample collection:
Various clinical specimens can be used for NAATs, including sputum, bronchoalveolar lavage fluid, cerebrospinal fluid,
pleural fluid, and tissue samples. The choice of sample depends on the site of infection and the type of NAAT being
used. It is crucial to follow proper sample collection and handling procedures to ensure accurate and reliable results.
• Test procedure:
The specific steps of a NAAT may vary depending on the particular method or test kit used. However, the general
workflow involves the following key steps:
1. Sample processing: the collected clinical sample is processed to extract the nucleic acids, typically DNA, from
the mycobacterial cells. This step involves the disruption of the bacterial cells and the purification of the
extracted DNA to remove inhibitors that may interfere with the amplification process.
2. Target amplification: the extracted DNA is subjected to amplification using a technique called polymerase
chain reaction (PCR) or its variants, such as real-time PCR or reverse transcription PCR (RT-PCR). These
techniques enable the exponential amplification of specific target sequences if they are present in the sample.
The amplification step allows for the detection and quantification of the M. tuberculosis DNA.
3. Detection: the amplified DNA is detected using various methods, such as fluorescent probes, molecular
beacons, or DNA hybridization. These methods allow for the specific identification of the amplified target
sequences. Real-time PCR enables the monitoring of the amplification process in real-time, providing
quantitative data on the amount of target DNA present in the sample.
• Diagnosis of tuberculosis (NAATs)

23. Rapid molecular tests of diagnosing tuberculosis (TB) refer to newer diagnostic techniques that offer faster turnaround times and quicker results
compared to traditional diagnostic methods. These methods play a crucial role in expediting TB diagnosis, enabling prompt initiation of treatment and
reducing the transmission of the disease.
• Rapid molecular tests of diagnosing tuberculosis-1
GeneXpert MTB/RIF is a highly innovative molecular diagnostic assay used for the rapid detection of
Mycobacterium tuberculosis (MTB) and the assessment of rifampicin resistance, which is an indicator
of multidrug-resistant tuberculosis (MDR-TB).
• Principle:
The GeneXpert MTB/RIF assay is based on the principle of real-time polymerase chain reaction (PCR)
technology. It utilizes a fully automated cartridge system that integrates nucleic acid extraction,
amplification, and detection processes in a single device.
• Sample collection:
The assay can be performed on various clinical samples, including sputum, bronchoalveolar lavage
fluid, cerebrospinal fluid, and tissue samples. A good-quality sputum sample is often recommended as it
contains a higher concentration of MTB bacilli.
• Rifampicin resistance detection:
In addition to detecting the presence of MTB, the GeneXpert MTB/RIF assay also assesses rifampicin
resistance. Rifampicin is a key first-line anti-TB drug, and resistance to rifampicin is strongly
associated with MDR-TB. The assay specifically targets the rpoB gene, which is responsible for
encoding the beta subunit of the RNA polymerase enzyme in MTB. Mutations in the rpoB gene are
indicative of rifampicin resistance.
• Results and interpretation:
The GeneXpert MTB/RIF assay provides qualitative results regarding the presence or absence of MTB
and rifampicin resistance. The results are automatically interpreted by the GeneXpert instrument,
which displays the findings on a computer screen. The output may include information such as "MTB
detected," "MTB not detected," "rifampicin resistance detected," or "rifampicin resistance not detected."
GENEXPERT MTB/RIF

24. GenoType MTBDRplus is a molecular diagnostic assay used for the detection of drug
resistance in Mycobacterium tuberculosis (MTB). It specifically targets genetic mutations
associated with resistance to first-line anti-TB drugs, namely rifampicin (RIF) and
isoniazid (INH).
• Principle:
The GenoType MTBDRplus assay is based on the principle of DNA strip technology, also
known as line probe assay (LPA). It utilizes DNA probes immobilized on a nitrocellulose
strip to detect specific genetic mutations in the target genes associated with drug
resistance.
• Target genes:
The assay targets specific regions of the MTB genome that are known to contain mutations
associated with resistance to rifampicin and isoniazid. For rifampicin resistance, it detects
mutations in the rpoB gene, which codes for the beta subunit of the RNA polymerase
enzyme. For isoniazid resistance, it detects mutations in the katG gene and the inhA
promoter region.
• Sample collection:
The GenoType MTBDRplus assay can be performed on various clinical specimens,
including sputum, culture isolates, or smear-positive samples. The choice of sample
depends on the availability and quality of the specimen.
• Interpretation of results:
The interpretation of results is based on the presence or absence of specific bands or dots
on the nitrocellulose strip. The assay provides information on rifampicin and isoniazid
resistance, as well as susceptibility to these drugs. The absence of a wild-type band and the
presence of a mutant band indicate the presence of drug resistance-associated mutations.
GenoType MTBDRplus
Results for patterns of Genotype MTBDR plus strips
№1 – Negative control.
№2 – INH monoresistant (inhAC15T mutation).
№3 – MDR TB (rpoB mutation in 530–533 region, katG S315T1 mutation).
№4 – MDR (rpoB S531L, katG S315T1 mutation).
№5 – MDR (rpoB S531L, inhAC15T mutation).
№6 – INHmono-resistant (inhAC15T mutation).
№7 – MDR (rpoB S531L, katG S315T1 mutation).
• Rapid molecular tests of diagnosing tuberculosis-2

25. • Rapid molecular tests of diagnosing tuberculosis-3
*(a) Mutations in other genes can result in resistance to thionamides. Consequently, absence of inhA
mutation does not rule out resistance.
*(b) Specific mutations in gyrA (e.g. mutations recognized by the probes MUT3B, 3C, 3D) are associated
with high-level fluoroquinolones resistance.
RMTs have a good specificity, but are less sensitive than culture. Their various levels of complexity
determine their use at different levels of health facilities. Low complexity RMTs are preferred in routine
practice.
Main performances of Xpert assays

26. I. Risk groups on social factors:
• homeless;
• migrants and refugees;
• the prisoners and persons released from places of detention and members
of their families;
• needy persons;
• unemployed;
• persons who are on the account in the employment service;
• the persons who addressed to department of subsidies;
• persons in receivers distributors;
• persons, in temporary detention centers
• Risk groups for tuberculosis-1

27. II. Risk groups on medicobiological factors:
• patients with a diabetes mellitus ;
• patients with a silicosis;
• patients who it is long were treated by corticosteroids;
• patients receiving immunosuppressive and radiation therapy;
• cancer patients;
• HIV-positive people and patients with AIDS;
• persons with serious chronic somatic illnesses;
• the persons abusing alcohol;
• the persons abusing drugs;
• the patients who are in narcological and mental health facilities;
• persons with residual changes after spontaneously cured tuberculosis;
• persons who recovered from tuberculosis;
• all X-ray positive person.
III. Risk groups on epidemic factors:
• contact with TB patients and members of their families.
• Risk groups for tuberculosis-2

28. Thank you
for your attention!