1. Tuberculosis DIAGNOSIS
Treatment Action Group
TB/HIV Advocacy Toolkit
September 2017
With thanks to Adam Almeida and Andolyn Medina

2. Topics to be covered Fundamentals: what is “diagnosis”?
Active TB Diagnosis
Screening
Tools for diagnosis: microbiological confirmation
Tools for diagnosis: LAM
Diagnosing active TB in special populations
Latent TB Infection
Tools for diagnosis
The Main Points

3. FUNDAMENTALS: WHAT IS DIAGNOSIS?

4. What is DIAGNOSIS?
Diagnosis is the evaluation of a health outcome (e.g., active TB disease or latent TB infection), using a method or tool to measure, identify, or analyze conditions of the body
in vitro diagnostics use samples taken from the human body– most TB diagnostics are in vitro
in vivo diagnostics are conducted in the body—the tuberculin skin test used for diagnosing latent TB infection is an in vivo diagnostic
Diagnostic tests detect changes in our bodies that indicate an unhealthy state or look for the source of disease
TB diagnostics are used to:
identify latent TB infection
find active TB disease
provide information about drug resistance (what drugs will work best for treatment)
FUNDAMENTALS

5. TB Diagnostic Vocabulary
Sensitivity: refers to the proportion of people with a disease that are correctly identified by a diagnostic tool as having the condition
Specificity: refers to the proportion of people without a disease that are correctly identified by a diagnostic tool as not having the condition
False Positive: a test that incorrectly shows someone as having the health outcome of interest (TB, TB infection, drug resistance) when they do not
This shows that a test has low specificity
False Negative: a test that incorrectly shows someone as not having the health outcome of interest (TB, TB infection, drug resistance) when they do
This shows that a test has low sensitivity
FUNDAMENTALS

6. TB Diagnostic Vocabulary
Media: the substances (solid or liquid) containing nutrients that allow TB bacteria to be grown in a laboratory setting
Culture: the process of growing TB bacteria on a substance that provides nutrients (food) to allow the TB to grow
Drug susceptibility testing (DST): a test to detect drug resistance– that is, a test to see which drugs will work best against a particular strain of TB
Gold standard: the best way to do something
FUNDAMENTALS

7. ACTIVE TB DIAGNOSIS

8. WHAT IS SCREENING?
Screening is used to detect possible cases of TB among vulnerable populations at high risk for contracting the disease
Screening tools are typically not very specific, but they are sensitive
This means that false positives are common, but false negatives are rare
Screening is used to rule out active cases of TB or to continue on with microbiological testing
Not everyone needs to be tested for TB, efforts should be focused on high-risk groups (outlined in the next slide)
The two main screening tools for TB are symptom screening and chest X-ray
SCREENING

9. SYMPTOM SCREENING
Symptom screening can be performed in groups at high risk for contracting TB
Symptoms of TB:
Cough
Hemoptysis (coughing up blood)
Fever
Night sweats
Weight loss
High-risk for TB:
People with close contact with someone with TB
People with HIV
Workers exposed to crystalline silica dust
Disadvantages
Inconclusive (not a final answer)
Occurs when disease has already progressed
Not useful for extrapulmonary TB (TB outside the lungs)
Potential to increase number of false positives
Advantages
Helps people seek healthcare
Allows the diagnostic process to start
Inexpensive
Source:
SCREENING

10. CHEST X-RAY
Chest radiography is another option for screening, it can be used together with symptom screening to best determine who should continue on with diagnostic testing
Advantages
Quick
High sensitivity
Inexpensive
Widely used
Disadvantages
Low specificity
Requires additional testing
Cannot be used for extrapulmonary TB
Active TB doesn’t always look the same (especially in people with HIV)
Abnormal Chest X-Ray
Adapted from Dr. Madhukar Pai
SCREENING

11. DIAGNOSTIC TECHNIQUES
Microbiological confirmation means diagnosing TB by detecting the presence of M. tuberculosis (MTB), the bacterium or bug that causes TB
Microbiological confirmation is the preferred way to diagnose active TB
There are three types of microbiological tests for TB:
1) See the bug
Sputum smear microscopy
2) Multiply the bug (multiply genetic information from MTB)
Nucleic Acid Amplification Test (NAAT)*
Xpert MTB/RIF ULTRA
Line Probe Assays
3) Grow the bug
Solid culture
Liquid culture (MGIT, MODS, BacT/ALERT 3D)
Concept courtesy of Dr. Madhukar Pai
*NOTE: TB LAMP, not to be confused with LAM (which is covered later in this deck) is omitted due to limited utility as compared to Xpert MTB/RIF Ultra
DIAGNOSTIC TOOLS

12. 1) SPUTUM SMEAR MICROSCOPY
Advantages:
Same-day results
Low tech
Inexpensive
Widely available
Can be used for treatment monitoring
Procedure
Collect sputum
Smear small volume of sputum on glass slide
Stain with acid fast stain (Ziehl-Neelsen)
Flood slide with methylene blue counterstain
Decolorize with acid-alcohol solution
Scan entire sample under 40x magnification
Confirm the presence or absence of MTB
Disadvantages
Low sensitivity (50%) among all cases
Only useful for TB in lungs
Not accurate for children, people with HIV
Doesn’t give info on drug susceptibility
Multi-step, complicated procedure
Can’t distinguish MTB from other related bacteria (non-TB mycobacteria)
results are reported as:
Sputum smear-positive TB (or acid fast bacilli-positive)- a single positive sputum smear in which MTB is identified
Sputum smear-negative TB (acid fast bacilli-negative) - no visible presence of a single MTB, but persistence of TB-like symptoms, chest X-ray images that are consistent with pulmonary TB, and the decision of the clinician to treat using anti-TB drugs. If available, culture test can be performed to grow and confirm MTB.
MTB
DIAGNOSTIC TOOLS

13. Optimization Of Microscopy
The WHO recommends the following strategies to improve microscopy:
Use fluorescence staining
Use light-emitting diode (LED) microscope
Inexpensive, battery-operated light source can be used
These additions make bacteria easier to detect, offering about 10% increase in sensitivity and making smear 25% faster compared to conventional microscopy
results are reported as:
Sputum smear-positive TB (or acid fast bacilli-positive)- a single positive sputum smear in which MTB is identified
Sputum smear-negative TB (acid fast bacilli-negative) - no visible presence of a single MTB, but persistence of TB-like symptoms, chest X-ray images that are consistent with pulmonary TB, and the decision of the clinician to treat using anti-TB drugs. If available, culture test can be performed to grow and confirm MTB.
BUT even still, smear is less sensitive and specific than Xpert MTB/RIF ULTRA, and gives no information on drug resistance
Image source: Health Biz Informa
DIAGNOSTIC TOOLS

14. 2) Nucleic acid amplification tests
Nucleic acid amplification testing (NAAT) searches the genetic information in the tests sample to find the presence of MTB
It can analyze two different kinds of genetic information: DNA and RNA
NAAT is able to analyze genetic mutations that cause drug resistance as well, allowing the test to determine which treatment would be most effective
NAAT is a fast testing method because the genetic information is extracted and amplified, rather than waiting for the bacteria to grow
DIAGNOSTIC TOOLS

15. GENEXPERT
GeneXpert system is a cartridge-based test to detect MTB and resistance to the drug rifampicin
The cartridge is called “Xpert MTB/RIF ULTRA”
The new Ultra cartridge is more sensitive than the previous Xpert MTB/RIF cartrdige, especially in people with HIV, people with extrapulmonary TB, and children
The system analyzes a sample by extracting MTB genetic information, multiplying it, and reading its genetic code
The WHO recommends GeneXpert as the initial test for all adults and children in need of TB evaluation
In the future…
A small, portable machine with only one module (can only run one test at a time) called Omni is coming out in 2018
A cartridge in development may be able to detect resistance to more drugs (isoniazid, fluoroquinolones, and the second-line injectables)
DIAGNOSTIC TOOLS

16. (Image source: Cepheid)
GeneXpert: Advantages and Disadvantages
Advantages:
One step process—automated
Quick (results in <2 hrs)
Requires fewer biosafety measures than culture/LPA, so can be used in lower-level laboratories
High sensitivity
High specificity
Can detect rifampicin resistance
Same machine can also be used for HIV, hepatitis C diagnoses/viral load monitoring
Can work on many extrapulmonary TB samples
Disadvantages:
Reliant on electricity
Expensive
Cannot be used to track treatment progress
Requires annual calibration
GeneXpert XVI
(Image source: Cepheid)
DIAGNOSTIC TOOLS

17. Line Probe Assay
Line probe assays (LPAs) are another genetic or molecular test
They can detect presence of MTB
First-line LPAs can detect rifampicin and isoniazid resistance (MDR-TB)
Second-line LPAs can be used to detect resistance to the second-line injectable drugs and fluoroquinolones
LPAs are very important for quickly guiding treatment decisions:
To detect resistance to isoniazid, which has worse health outcomes and is not detected by GeneXpert
To determine which people with MDR-TB can take the shortened regimen
DIAGNOSTIC TOOLS

18. Line Probe ASSAy Advantages: Disadvantages:
Can perform multiple tests at once
Quick (results in under 48 hrs)
Accurate
Only rapid test to give information about isoniazid resistance
Necessary for guiding treatment decisions
Disadvantages:
Cannot fully replace other methods, like conventional cultures
Not as fast as Xpert
Expensive
Requires well-trained staff in a professional laboratory
Has high biosafety requirements (can only be used in certain labs)
DIAGNOSTIC TOOLS

19. 3) CULTURE
Culture is considered the gold standard, meaning the most accurate test, for TB
Culture is the growth of bacteria in vitro (in a controlled environment outside of the body) on a medium (substance providing nutrients)
The number of colony forming units (CFU)—MTB bacterial cells that can grow—can then be counted, either visually or by automated detection
Culture positive: presence of any MTB CFUs
Culture negative: no CFU detected after 42 days
Advantages:
High sensitivity
High specificity
Ability to perform drug susceptibility testing (called “phenotypic testing”)
Can assess treatment progress
Disadvantages:
Requires trained staff
Automated liquid culture is expensive (but getting more affordable)
Solid culture takes longer
DIAGNOSTIC TOOLS

20. MODS
Microscopic observation drug susceptibility (MODS) is an inexpensive, non-commercial liquid culture option
It gives results within seven days
Due to its low cost, MODS is feasible in resource-limited areas
The WHO recommends MODS as a method of detection as countries move towards the roll-out of automated liquid culture methods
DIAGNOSTIC TOOLS

21. The rule of twos Test Unit of time GeneXpert 2 hours line probe assay
An easy way to remember how long each microbiological test takes is to remember the number 2:
Test
Unit of time
GeneXpert 2
hours
line probe assay
days
liquid culture
weeks
solid culture
months
DIAGNOSTIC TOOLS

22. POINT-OF-CARE TESTING
Most microbiological tests for detecting MTB provide the clearest diagnosis of TB and allow drug susceptibility to be tested as well
However, these tests have many limitations: they can be expensive, lengthy, and require infrastructure, like laboratories and trained staff, to be in place to analyze the specimens. Most importantly, current microbiological tests do not provide information at the point-of-care level
The LAM test is a newly introduced diagnostic tool that provides point-of-care TB testing in some people with HIV
DIAGNOSTIC TOOLS

23. THE LAM TEST
The lateral flow urine lipoarabinomannan (LAM) assay for use only in some people living with HIV
LAM is an antigen located in the cell walls of MTB
LAM is only present in people with active TB disease
High levels of LAM in people with immunosuppression from HIV
Simple, dipstick test (similar to a pregnancy test)
Low sensitivity, but still useful
In 2015, WHO recommended TB LAM for people with HIV with CD4<100/mm3 or who are seriously ill
Only test demonstrated to reduce TB deaths
Best introduced in hospitals in settings with high burdens of TB/HIV for anyone admitted with advanced HIV; can also be used in outpatient settings
Photo from Alere
Read more at
DIAGNOSTIC TOOLS

24. THE LAM TEST If positive, treatment should be started immediately
If negative, follow up with another kind of test since sensitivity is not very high
Advantages
Point-of-care test
Simple, very low-tech
Fast (25 minutes)
Inexpensive
Easy to collect and store urine
Detection in population that cannot use other diagnostic techniques
Does not require electricity or labs
Saves lives by allowing earlier treatment start*
Disadvantages
Test must be followed up with other diagnostics
Low sensitivity
No info on drug resistance
Used in limited patient population
Cannot distinguish MTB from other non-TB mycobacteria
Source:
*Peter, J. G. et al. (March 01, 2016). Effect on mortality of point-of-care, urine-based lipoarabinomannan testing to guide tuberculosis treatment initiation in HIV-positive hospital inpatients: a pragmatic, parallel-group, multicountry, open-label, randomised controlled trial. The Lancet, 387, 10024,
DIAGNOSTIC TOOLS

25. Diagnosing Extrapulmonary TB
Most TB tests rely on sputum as the sample, but this will not work for TB outside the lungs (extrapulmonary TB)
Usually performed due to clinical suspicion
Sample taken from the suspected site of disease
Options: need to use a combination of tests
Smear: likely to be negative
GeneXpert: on tissue biopsies, gastric contents, pus, cerebrospinal fluid, and urine
Culture: helpful, but takes 2-3 weeks
Biopsy: very helpful
If nothing works, treat TB based on clinical judgment even in absence of confirmation (empiric treatment)
No role for blood tests
Blood is not used in any active TB diagnosis
DIAGNOSTIC TOOLS
Slide courtesy of Dr. Madhukar Pai

26. DIAGNOSIS IN CHILDREN
Children are an especially vulnerable population for developing active TB
Diagnosing children comes with its own set of difficulties since:
Children have difficulty producing sputum samples
Children have lower levels of bacteria in the body
TB is often diagnosed in children without microbiological confirmation
Instead, a physician will use symptom screening, chest X-rays, or points of contact with infected individuals
For using microbiological diagnostic tests, GeneXpert is preferred
SPECIAL POPULATIONS

27. DRUG SUSCEPTIBILITY TESTING
Drug susceptibility testing is important for deciding which treatment plan would be best suited for the individual with TB
Drug-resistant strains of MTB occur are rising in prevalence due to inappropriate treatment regimens and poor compliance
LPA: line probe assay
DST: drug susceptibility testing
FQs: fluoroquinolones
INH: isoniazid
DIAGNOSTIC TOOLS

28. TOOLS FOR DIAGNOSIS: TB infection

29. Latent TB INFECTION DIAGNOSIS
Goal: prevent active TB by giving preventive therapy
Options:
Tuberculin Skin Test (TST)
Also referred to as the Mantoux test
Uses purified protein derivative (PPD)
Interferon Gamma Release Assays (IGRAs)
TB Platinum
T Spot.TB Test
QuantiFERON-TB Gold Plus
There is no “gold standard” for diagnosing latent TB infection
No test can predict who with latent TB infection will develop active TB disease
WHO recommends that TST be performed instead of IGRA in resource-poor settings, due to its cost and similar effectiveness
For people with HIV, do not need to diagnose LTBI to start preventive therapy, as long as active disease is ruled out
DIAGNOSING TB INFECTION

30. TubErculin Skin Test (TST)
Tuberculin Skin Test (TST) detects TB infection through the skin by exposing the skin to an antigen, causing a bump if an infection is present
Advantages
Simple, easy to administer
Inexpensive
Disadvantages
Requires refrigeration
Requires training to administer
Low specificity
Low sensitivity
Requires people to come back a different day for results
Cannot distinguish active TB from LTBI
DIAGNOSING TB INFECTION

31. Interferon Gamma Release Assays (IGRA)
IGRAs uses a blood sample to determine the body’s immune reactivity to MTB
White blood cells from people with MTB infection release interferon-gamma when they meet MTB antigens
Fresh blood samples are mixed with antigens and controls to compare reactions
Advantages:
One-time test
Quick (results in 24h)
Does not boost responses measured by subsequent tests
Prior BCG (bacille Calmette-Guérin) vaccination does not cause a false-positive result
Disadvantages:
Expensive
Blood must be processed quickly after collection
Errors arise easily
Cannot distinguish active TB from LTBI
Limited data on use in children and people with HIV
DIAGNOSING TB INFECTION
Source:

32. The main points

33. DIAGNOSTICS FUNDING, 2015 MAIN POINTS
Source:
MAIN POINTS

34. DIAGNOSTICS FUNDING, 2015 MAIN POINTS
Source:
MAIN POINTS

35. Limitations in Diagnosis
TB screening and diagnosis requires a combination of different methods and techniques to accurately test people
There is no singlr TB test that can do it all
Most TB diagnostic tests are not appropriate for use as point-of-care tests in local settings where most TB patients are seen
Culture and LPA require high biosafety levels to protect the workers and get accurate results.
Majority of TB tests require electricity, equipment, and some degree of infrastructure
Most are costly for national governments to afford without donor support
We need more investment in TB research and development to develop better tests
Note to facilitator: this is not an exhaustive list and can be used as start for discussion.
MAIN POINTS

36. How to know if a diagnostic is useful in your setting
Is the test sensitive and specific?
What kind of TB (e.g., smear-negative TB, drug resistant TB, latent TB) and populations (e.g., infants, people with HIV) is it useful for?
How long does the test take?
What does the test tell us (e.g. infection, disease)?
Does its use need a lot of technology and training?
What type of specimen does it need (e.g. sputum, urine)?
Does it need a high level of biosafety? (e.g. does it involve culturing the bacteria)?
What does it cost?
Has the test been studied objectively and endorsed by WHO?
Note to facilitator: this is not an exhaustive list and can be used as start for discussion.
MAIN POINTS

37. MAIN MESSAGE— All Countries need:
GeneXpert Xpert MTB/RIF ULTRA as the initial test for ALL people needing testing for TB
Line Probe Assay (both first- and second-line) to quickly guide treatment decisions
Liquid culture (MGIT) for full drug susceptibility testing AND monitoring drug-resistant TB treatment
Smear microscopy for monitoring standard TB treatment, but GeneXpert is the preferred test for diagnosing TB
In areas with high burdens of TB/HIV:
TB LAM (Determine TB LAM Ag) for quickly, easily finding TB in people very sick with HIV and starting them on TB treatment
MAIN POINTS

38. ADDITIONAL RESOURCES
Treatment Action Group (TAG) has created An Activist’s Guide to Tuberculosis Diagnostic Tools, which expands upon what has been outlined in these slide decks. To access: s/default/files/TB%20Diagnostics%20Gu ide.pdf TAG also issued An Activist's Guide to the TB LAM Test, available at: nt/activists-guide-tb-lam-test
MAIN POINTS