Tuberculosis DIAGNOSIS

Tuberculosis DIAGNOSIS Treatment Action Group TB/HIV Advocacy Toolkit July 2019 With thanks to Adam Almeida, Andolyn Medina, and Dr. Jennifer Furin

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

FUNDAMENTALS: WHAT IS DIAGNOSIS?

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

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

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

ACTIVE TB DIAGNOSIS

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

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: http://www.who.int/tb/publications/tbscreening_factsheet.pdf?ua=1 SCREENING

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

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

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) MTB DIAGNOSTIC TOOLS

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 • 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

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

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 cartridge, 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 • A cartridge in development will be able to detect resistance to more drugs (isoniazid, fluoroquinolones, and the second-line injectables) DIAGNOSTIC TOOLS http://apps.who.int/iris/bitstream/10665/254792/1/WHO-HTM-TB-2017.04-eng.pdf

GeneXpert: Advantages and Disadvantages • Disadvantages: • Reliant on electricity • Expensive • Cannot be used to track treatment progress • Requires annual calibration • 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 GeneXpert XVI (Image source: Cepheid) DIAGNOSTIC TOOLS

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

Line Probe ASSAy • Advantages: • 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

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 • Disadvantages: • Requires trained staff • Automated liquid culture is expensive (but getting more affordable) • Solid culture takes longer • Advantages: • High sensitivity • High specificity • Ability to perform drug susceptibility testing (called “phenotypic testing”) • Can assess treatment progress DIAGNOSTIC TOOLS

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

The rule of twos An easy way to remember how long each microbiological test takes is to remember the number 2: DIAGNOSTIC TOOLS

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, currentmicrobiological 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

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 • Newer, more sensitive version of the LAM known as “FujiLAM” may improve the performance and utility of this type of test • 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 http://treatmentactiongroup.org/content/activists-guide-tb-lam-test DIAGNOSTIC TOOLS

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: http://www.who.int/tb/areas-of-work/laboratory/policy_statement_lam_web.pdf *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, 1187-1197. DIAGNOSTIC TOOLS

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 • 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 usedin any active TB diagnosis DIAGNOSTIC TOOLS Slide courtesy of Dr. Madhukar Pai

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

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 are rising in prevalence due to inappropriate treatment regimens and poor compliance Xpert MTB/RIF Ultra used as initialdiagnostic rifampicin sensitive rifampicin resistant Startall-oral MDR-TB treatmentregimenwithgroup A and B drugs, sendforsecond-line LPA and liquidculture/DST Start standard, first-line therapy, send for LPA sensitive to FQs and injectables resistant to INH INH sensitive resistant to FQs Continue all-oral MDR-TB treatment regimen Add Group C drugs (i.e. delamanid), consider extension of BDQ or DLM beyond 6 months Asses for risk of amplification of rifampicin resistance . If no risk, tart levofloxacin and continue with RIF, EMB and PZA daily for 6 months Continue standard first-line therapy • LPA: line probe assay • DST: drug susceptibility testing • FQs: fluoroquinolones • INH: isoniazid • BDQ: bedaquiline • DLM: delamanid DIAGNOSTIC TOOLS

Eye on the Future: Newer Tests for TB • Newer NAAT tests include the Xpert XDRTB test which can assess for resistance to isoniazid, levofloxacin, and kanamycin; TrueNAT test which may be more easily used at “point of care” • FujiLAM may offer improved sensitivity for urine-based TB testing • Whole genome sequencing is a test that looks at all the DNA of MTB to see if there are any changes or mutations that could lead to drug resistance • All of these tests likely to be used more in the future

TOOLS FOR DIAGNOSIS: TB infection

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 and children under five, do notneed to diagnose LTBI to start preventive therapy, as long as active disease is ruled out DIAGNOSING TB INFECTION

TubErculin Skin Test (TST) • Tuberculin Skin Test (TST) detects TB infection through the skinby 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

Interferon Gamma Release Assays (IGRA) • IGRAs uses a blood sampleto determine the body’s immune reactivity to MTB • White blood cells from people with MTB infection release interferon-gammawhen 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: http://www.cdc.gov/tb/publications/factsheets/testing/igra.htm

The main points

DIAGNOSTICS research FUNDING, 2017 MAIN POINTS Source: http://www.treatmentactiongroup.org/tbrd2018

TB RESEARCH FUNDING, 2017 RESEARCH Source: http://www.treatmentactiongroup.org/tbrd2017

Limitations in Diagnosis • TB screening and diagnosis requires a combination of different methods and techniques to accurately test people • There is no single TB test that can do it all • Most TB diagnostic tests are not well-suited for use as point-of-caretests in local settings where most TB patients are seen • Culture and LPA require high biosafety levelsto protect the workers and get accurate results • Majority of TB tests require electricity, equipment, and some degree of infrastructure • Most are costlyfor national governments to afford without donor support • We need more investment in TB research and development to develop better tests MAIN POINTS

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? MAIN POINTS

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

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: http://www.treatmentactiongroup.org/sites/default/files/TB%20Diagnostics%20Guide.pdf • TAG also issued TB LAM Testing Briefs, available at:http://treatmentactiongroup.org/content/tb-lam-testing-briefs-2019 MAIN POINTS

Tuberculosis DIAGNOSIS