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Targeted Testing and Treatment of Latent Tuberculosis Infection


Slide 1: Targeted Tuberculosis (TB) Testing and Treatment of Latent Tuberculosis Infection

December 2011
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Division of Tuberculosis Elimination

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Slide 2: Targeted TB Testing and Treatment of Latent TB Infection

  • As TB disease rates in the United States decrease, finding and treating persons at high risk for latent TB infection (LTBI) has become a priority.
  • Targeted TB testing is used to focus program activities and provider practices on groups at the highest risk for TB.
  • Treatment of LTBI substantially reduces the risk that persons infected with M. tuberculosis will progress to TB disease. 

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Slide 3: Latent TB Infection (LTBI)

LTBI is the presence of M. tuberculosis organisms (tubercle bacilli) without signs and symptoms or radiographic or bacteriologic evidence of TB disease.

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Slide 4: LTBI vs. Pulmonary TB Disease – 1

Latent TB Infection

  • Positive TST* or IGRA† result
  • Chest radiograph normal

Pulmonary TB Disease

  • TST or IGRA is usually positive
  • Chest radiograph is usually abnormal

*tuberculin skin test
† Interferon-Gamma Release Assay

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Slide 5: LTBI vs. Pulmonary TB Disease – 2

Latent TB Infection

  • No symptoms or physical findings suggestive of TB disease
  • If done, respiratory specimens are smear and culture negative

Pulmonary TB Disease

  • Symptoms may include one or more of the following: fever, cough, night sweats, weight loss, fatigue, hemoptysis, decreased appetite
  • Respiratory specimens are usually culture  positive (smear positive in about 50% of patients)

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Slide 6: Targeted TB Testing

  • Essential TB prevention  and control strategy
  • Detects persons with LTBI who would benefit from treatment
  • De-emphasizes testing of groups that are not at high risk for TB
  • Can help reduce the waste of resources and prevent inappropriate treatment

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Slide 7: Treatment of LTBI – Milestones – 1

For more than 3 decades, an essential component of TB prevention and control in the United States has been the treatment of persons with LTBI to prevent TB disease.

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Slide 8: Treatment of LTBI – Milestones – 2

1965: American Thoracic Society (ATS) recommends treatment of LTBI for those with previously untreated TB, tuberculin skin test (TST) converters, and young children.

1967: Recommendations expanded to include all TST positive reactors (≥ 10 mm).

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Slide 9: Treatment of LTBI – Milestones – 3

1974: CDC and ATS guidelines established for pretreatment screening to decrease risk of hepatitis associated with treatment

Treatment recommended for persons ≤ 35 years of age

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Slide 10: Treatment of LTBI – Milestones – 4

1983: CDC recommends clinical and laboratory monitoring of persons ≥ 35 who require treatment for LTBI

1998: CDC recommends 2 months of rifampin (RIF) plus pyrazinamide (PZA) as an option for HIV-infected patients (later changed)

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Slide 11: Treatment of LTBI – Milestones – 5

2000: CDC and ATS issue updated guidelines for targeted testing and LTBI treatment1

9-month regimen of isoniazid (INH) is preferred

2-month regimen of RIF and PZA and a 4 month regimen of RIF recommended as options (later changed)

1 MMWR June 9, 2000; 49(No. RR-6)

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Slide 12: Treatment of LTBI – Milestones – 6

2001: Owing to liver injury and death associated with 2-month regimen of RIF and PZA, use of this option de-emphasized in favor of other regimens2

2003: 2-month regimen of RIF and PZA generally not recommended — to be used only if the potential benefits outweigh the risk of severe liver injury and death3

2MMWR August 31,  2001; 50(34): 733-735

3MMWR August 8,  2003; 52(31): 735-739

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Slide 13: Treatment of LTBI – Milestones – 7

2011:  CDC recommends  12-doses (3 months) of isoniazid (INH) and rifapentine (RPT) as an option equal to the standard 9-month  INH regimen for certain groups*

*Recommendations for Use of an Isoniazid–Rifapentine Regimen with Direct Observation to Treat Latent Mycobacterium tuberculosis Infection

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Slide 14: Identifying Risk Factors That Lead to Development of TB Disease

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Slide 15: Persons at Risk for Developing TB Disease

Persons at high risk for developing TB disease fall into 2 categories:

  • Those who have an increased likelihood of exposure to persons with TB disease
  • Those with clinical conditions that increase their risk of progressing from LTBI to TB disease

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Slide 16: Increased Likelihood of Exposure to Persons with TB Disease

Persons at risk for exposure to persons with TB disease include:

  • Close contacts to person with infectious TB
  • Residents and employees of high-risk congregate settings (e.g., correctional facilities, homeless shelters, health care facilities)
  • Recent immigrants from TB-endemic regions of the world (within 5 years of arrival to the United States)

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Slide 17: Increased Risk for Progression to TB Disease – 1

Persons more likely to progress from LTBI to TB disease include:

  • HIV-infected persons
  • Those with a history of prior, untreated TB or fibrotic lesions on chest radiograph
  • Children ≤ 5 years with a positive TST

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Slide 18: Increased Risk for Progression to TB Disease – 2

Persons more likely to progress from LTBI to TB disease include:

  • Underweight or malnourished persons
  • Injection drug users
  • Those receiving TNF-α antagonists for treatment of rheumatoid arthritis or Crohn’s disease

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Slide 19: Increased Risk for Progression to TB Disease – 3

Persons more likely to progress from LTBI to TB disease include:

  • Those with certain medical conditions such as:
    • Silicosis
    • Diabetes mellitus
    • Chronic renal failure or on hemodialysis
    • Solid organ transplantation (e.g., heart, kidney)
    • Carcinoma of head or neck
    • Gastrectomy or jejunoilial bypass

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Slide 20: Testing for M. tuberculosis Infection

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Slide 21: Testing for M. tuberculosis Infection

  • There are two testing methods available for the detection of M. tuberculosis  infection in the United States:
    • Mantoux tuberculin skin test (TST)
    • Interferon-gamma release assays (IGRA)
  • These tests do not exclude LTBI or TB disease
  • Decisions about medical and public health management should include other information, and not rely only on TST or IGRA results

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Slide 22: Mantoux Tuberculin Skin Test

Skin test that produces delayed-type hypersensitivity reaction in persons with M. tuberculosis  infection

  •  TST is useful for:
    • Determining how many people in a group are infected (e.g., contact investigation)
    • Examining persons who have symptoms of TB disease
  • Multiple puncture tests (e.g., Tine Test) are inaccurate and not recommended

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Slide 23: Administering the TST

  • Inject 0.1 ml of 5 TU PPD tuberculin solution intradermally on volar surface of lower arm using a 27-guage needle
  • Produce a wheal 6 to 10 mm in diameter

[Image: Drawing of gloved hands holding a tuberculin syringe and injecting PPD into an arm. The drawing shows a discrete, pale elevation of the skin (wheal) has been formed.]

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Slide 24: Reading the TST – 1

  • Measure reaction in 48 to 72 hours
  • Measure induration, not erythema
  • Record reaction in millimeters,  not “negative” or “positive”
  • Ensure trained health care professional measures and interprets the TST

[Image: Drawing of a hand holding a TST ruler and measuring the diameter of an induration. Only the induration is being measured; this is the correct way to read the TST reaction.]

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Slide 25: Reading the TST – 2

  • Educate patient and family regarding significance of a positive TST result
  • Positive TST reactions can be measured accurately for up to 7 days
  • Negative reactions can be read accurately for only 72 hours

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Slide 26: TST Interpretation – 1

≥ 5 mm induration is interpreted as positive in

  • HIV-infected persons
  • Close contacts to an infectious TB case
  • Persons with chest radiographs consistent with prior untreated TB

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Slide 27: TST Interpretation – 2

≥ 5 mm induration is interpreted as positive in

  • Organ transplant recipients
  • Other immunosuppressed patients (e.g. , those taking the equivalent of > 15 mg/d of prednisone for 1 month or those taking TNF-α antagonists)

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Slide 28: TST Interpretation – 3

≥ 10 mm induration is interpreted as positive in

  • Recent immigrants
  • Injection drug users
  • Residents or employees of congregate settings
  • Mycobacteriology laboratory personnel

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Slide 29: TST Interpretation – 4

≥ 10 mm induration is interpreted as positive in

  • Persons with clinical conditions that place them at high risk
  • Children < 4 years; infants, children, and adolescents exposed to adults at high-risk

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Slide 30: TST Interpretation – 5

≥ 15 mm induration is interpreted as positive in

  • Persons with no known risk factors for TB.
    • Although skin testing programs should be conducted only among high-risk groups, certain individuals may require TST for employment or school attendance.  Diagnosis and treatment of LTBI should always be tied to risk assessment.

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Slide 31: Factors That May Cause False-Positive TST Reactions

  • Nontuberculous mycobacteria
    • Reactions caused by nontuberculous mycobacteria are usually £10 mm of induration
  •   BCG vaccination
    • Reactivity in BCG vaccine recipients generally wanes over time; positive TST result is likely due to TB infection if risk factors are present

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Slide 32: Factors That May Cause False-Negative TST Reactions -1

  •    Anergy
    • Inability to react to a TST because of a weakened immune system
    • Usefulness of anergy testing in TST-negative persons who are HIV infected has not been demonstrated

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Slide 33: Factors That May Cause False-Negative TST Reactions – 2

  •   Recent TB Infection
    • Defined as less than 10 weeks after exposure
  •   Very young age
    • Newborns (< 6 months)

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Slide 34: Factors That May Cause False-Negative TST Reactions – 3

  •   Live virus vaccination
    • For example, measles or smallpox
    • Can temporarily suppress TST reactivity
  •   Overwhelming TB Disease
  •   Poor TST administration technique
    • For example, TST injection too shallow or too deep, or wheal is too small

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Slide 35: Boosting

  • Some people with LTBI may have a negative skin test reaction when tested years after infection because of a waning response.
  • An initial skin test may stimulate (boost) the ability to react to tuberculin.
  • Positive reactions to subsequent tests may be misinterpreted as new infections rather than “boosted” reactions.

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Slide 36: Two-Step Testing – 1

  • A strategy to determine the difference between boosted reactions and reactions due to recent infection.
    • If 1st test positive, consider infected; if negative, give 2nd test 1–3 weeks later
    • If 2nd test positive, consider infected; if negative, consider uninfected
  • Use two-step tests for initial baseline skin testing of adults who will be retested periodically (e.g., health care workers).

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Slide 37: Two-step TST Testing – 2

This is a diagram describing two-step TST testing. Administer a baseline skin test. If the reaction is positive, the person probably has TB infection; provide follow-up for positive TST and evaluate for LTBI treatment. If the reaction is negative, retest 1 to 3 weeks later. If the reaction to the second test is positive, it is considered a boosted reaction (due to TB infection that occurred a long time ago). Note: The person does have LTBI; a decision must be made whether to treat or not. Provide follow-up for positive TST and evaluate for LTBI treatment. If the reaction to the second test is negative, the person probably does not have TB infection. Repeat TST at regular intervals; a positive reaction could be due to a recent TB infection.

Slide 38: Interferon-Gamma Release Assays (IGRAs)

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Slide 39: Interferon-Gamma Release Assays (IGRAs)

  • Whole-blood test used to detect M.  tuberculosis  infection
  • Two U.S. Food and Drug Administration (FDA) approved IGRAs are commercially available in the U.S.:
    • QuantiFERON® -TB Gold-in-tube test (QFT-GIT)
    • T.SPOT®.TB test (T-Spot)

[Image: photo of QuantiFERON-TB Gold In-Tube test kit]

[Image: photo of T-Spot.TB test kit]

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Slide 40: How IGRAs Work – 1

  • Blood test that measures and compares amount of interferon-gamma (IFN-g) released by blood cells in response to antigens
  • Entails mixing blood samples with antigens from M. tuberculosis and controls

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Slide 41: How IGRAs Work – 2

  • Cells that recognize the antigen release  interferon-g
  • Amount of interferon released in response to M. tuberculosis  antigens is compared to amount released in response to other antigens

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Slide 42: Administering IGRAs

  • Confirm and arrange for delivery of blood sample within specific time-frame to ensure viability of blood samples
  • Draw blood sample according to test manufacturer’s instructions
  • Schedule a follow up appointment to receive test results, medical evaluation and possible treatment if needed

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Slide 43: Interpretation of IGRA Test Results

QFT-GIT test results are reported as positive, negative, indeterminate

T-Spot test results are reported as positive, negative, indeterminate, or borderline

Note: Laboratory should provide both quantitative and qualitative results

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Slide 44: Advantages of IGRAs

  • Requires a single patient visit to conduct  test
  • Results can be available within 24 hours
  • Does not boost responses measured by subsequent tests
  • Prior BCG vaccination does not cause false-positive IGRA test result

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Slide 45: Disadvantages/Limitations of IGRAs – 1

  • Errors in collecting and transporting blood, or  in  interpreting assays can decrease accuracy of IGRAs
  • Limited data on use of IGRAs to predict who will  progress to TB disease in the future

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Slide 46: Disadvantages/Limitations of IGRAs – 2

  • Tests may be expensive
  • Limited data on the use of IGRAS for
    • Children < 5 years of age;
    • Persons recently exposed to M. tuberculosis;
    • Immunocompromised persons; and
    • Serial testing

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Slide 47: TB Test Selection

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Slide 48: Selecting a Test to Detect TB Infection – 1

  •   IGRAs are preferred method of testing for
    • Groups of people who have poor rates of returning to have TST read
    • Persons who have received BCG vaccine
  • TST is the preferred method of testing for
    • Children under the age of 5

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Slide 49: Selecting a Test to Detect TB Infection – 2

Before initiating treatment for LTBI

  • Either TST or IGRA can be used without  preference for other groups that are tested for LTBI
  • Routine testing with TST and IGRA is NOT recommended

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Slide 50: Evaluation of Persons with Positive TB Test Results

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Slide 51: Evaluation of Persons with Positive TB Test Results

This is a diagram explaining the evaluation of persons with positive TB test results. If a person has a positive test result for TB infection and TB disease is ruled out, consider the person for treatment of LTBI. If the person accepts and is able to receive treatment for LTBI, the clinician should develop a plan of treatment with the patient to ensure the patient’s adherence. But if the person refuses or is unable to receive treatment for LTBI, then follow-up TSTs or IGRAs and serial chest radiographs are unnecessary; in that case, the clinician would educate the patient about the signs and symptoms of TB disease.

Slide 52: LTBI Treatment regimens 

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Slide 53: Initiating Treatment

Before initiating treatment for LTBI

  • Rule out TB disease by history, physical examination, chest radiography and, when indicated, bacteriologic studies
  • Determine prior history of treatment for LTBI or TB disease
  • Assess risks and benefits of treatment
  • Determine current and previous drug therapy

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Slide 54: Treatment Regimens for Latent TB Infection

Drug(s)

Duration

Interval

Minimum Doses

Isoniazid (INH)

9 months

Daily

270

Twice weekly

76

6 months

Daily

180

Twice weekly

52

Isoniazid (INH)and Rifapentine (RPT)

3 months

Once weekly

12

Rifampin (RIF)

4 months

Daily

120

Note: Rifampin (RIF) and Pyrazinamide (PZA) should not be offered to persons with LTBI.  RIF and PZA should continue to be administered in multidrug regimens for the treatment of persons with TB disease.

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Slide 55: Latent TB Infection Treatment Regimens – Isoniazid (INH) – 1

  • 9-month regimen of isoniazid (INH) is one of the preferred regimens
    • 6-month regimen is less effective but may be used if unable to complete 9 months
  • May be given daily or intermittently (twice weekly)
  • Use directly observed therapy (DOT) for intermittent regimen
  • Preferred regimen for children 2-11 years of age

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Slide 56: Latent TB Infection Treatment Regimens – Isoniazid (INH) – 2

  • Doses
    • INH daily for 9 months - 270 doses within 12 months
    • INH twice/week for 9 months - 76 doses within 12 months
    • INH daily for 6 months - 180 doses within 9 months
    • INH twice/week for 6 months - 52 doses within 9 months

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Slide 57: Latent TB Infection Treatment Regimens – Isoniazid (INH) and Rifapentine (RPT) – 1

  • 3-month regimen of INH and RPT is an option equal to 9-month INH regimen for treating LTBI in certain groups, such as otherwise healthy people, 12 years of age and older, who were recently in contact with infectious TB or who had tuberculin skin test conversions or positive blood test for TB*
  • Must use directly observed therapy (DOT)

*Recommendations for Use of an Isoniazid–Rifapentine Regimen with Direct Observation to Treat Latent Mycobacterium tuberculosis Infection

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Slide 58: Latent TB Infection Treatment Regimens – Isoniazid (INH) and Rifapentine (RPT) – 2

  • Not recommended for children younger than 12 years of age, HIV-infected people taking antiretroviral therapy, pregnant women,  or women expecting to be pregnant within the 12-week regimen
  • INH and RPT once a week for 3 months - 12 doses within 4 months

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Slide 59: Latent TB Infection Treatment Regimens – Rifampin

  • Rifampin (RIF) given daily for 4 months is an acceptable alternative when treatment with INH is not feasible.
  • In situations where RIF cannot be used (e.g., HIV-infected persons receiving protease inhibitors), rifabutin may be substituted.
  • RIF daily for 4 months - 120 doses within 6 months

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Slide 60: Latent TB Infection Treatment Regimens for Specific Situations – HIV-Infected Persons

HIV-Infected Persons

  • Consult an expert in managing HIV and TB
  • INH daily for 9-mo, rather than 6-mo, is optimal: 270 doses within 12 months
  • RIF is generally contraindicated for persons taking protease inhibitors or delavirdine
  • Rifabutin with dose adjustments can sometimes be substituted for RIF
  • INH/RPT regimen not recommended for HIV-infected people taking antiretroviral therapy

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Slide 61: Latent TB Infection Treatment Regimens for Specific Situations – Fibrotic Lesions

Persons with Fibrotic Lesions Suggesting Previous TB

  • Should be treated for LTBI if they have
    • A positive TST reaction (at least 5 mm) or IGRA result
    • No symptoms of infectious TB disease
    • No history of treatment for TB disease
  • Treat only after active disease excluded with sputum testing
  • Acceptable regimens include
    • 9 months of INH
    • 4 months of RIF (with or without INH)
    • 3 months of INH and RPT (12-dose regimen)

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Slide 62: Latent TB Infection Treatment Regimens for Specific Situations – Multidrug-Resistant TB

Contacts of Persons with Multidrug-Resistant TB

  • Consider risk for progressing to MDR disease before recommending LTBI treatment
  • When prescribing treatment for these contacts, consult an MDR TB expert

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Slide 63: Latent TB Infection Treatment Regimens for Specific Situations - Pregnancy

Pregnancy and Breast-Feeding

  • 9 months of INH daily or twice weekly; give with vitamin B6
  • If cannot take INH, consult with TB expert
  • Women at high risk for progression to TB disease should not delay LTBI treatment; monitor carefully
  • Breast-feeding not contraindicated

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Slide 64: Completion of Therapy

Completion of therapy is based on the total number of doses administered, not on duration alone.

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Slide 65: Management of Patient Who Missed Doses

  • Extend or re-start treatment if interruptions were frequent or prolonged enough to preclude completion
  • When treatment has been interrupted for more than 2 months, patient should be examined to rule out TB disease
  • Recommend and arrange for DOT as needed

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Slide 66: Monitoring Drug Treatment

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Slide 67: Clinical Monitoring – 1

Instruct patient to report signs and symptoms of adverse drug reactions:

  • Fever
  • Headache
  • Rash
  • Anorexia, nausea, vomiting, or abdominal pain in right upper quadrant
  • Fatigue or weakness
  • Dark urine
  • Persistent numbness in hands or feet

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Slide 68: Clinical Monitoring – 2

Monthly visits should include a brief physical exam and a review of:

  • Rationale for treatment
  • Adherence with therapy
  • Symptoms of adverse drug reactions
  • Plans to continue treatment

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Slide 69: Clinical Monitoring – 3

  • Incidence of hepatitis in persons taking INH is lower than previously thought (as low as 0.1%)
  • Hepatitis risk increases with age
    • Uncommon in persons < 20 years old
    • Nearly 2% in persons 50 to 64 years old
  • Risk increases with underlying liver disease or heavy alcohol consumption

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Slide 70: Laboratory Monitoring – 1

Baseline liver function tests (e.g., AST, ALT, and bilirubin) are not necessary except for patients with risk factors:

  • HIV infection
  • History of liver disease
  • Regular alcohol use
  • Pregnancy or in early postpartum period

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Slide 71: Laboratory Monitoring – 2

Repeat laboratory monitoring if patient has:

  • Abnormal baseline results
  • Current or recent pregnancy
  • High risk for adverse reactions
  • Symptoms of  adverse reaction
  • Liver enlargement or tenderness during examination

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Slide 72: Laboratory Monitoring – 3

  • Asymptomatic elevation of hepatic enzymes seen in 10%-20% of people taking INH
    • Levels usually return to normal after completion of therapy
  • Discontinue treatment  if transminase level exceeds 3 times the upper limit of normal if patient has symptoms of hepatoxicity, and 5 times the upper limit of normal if patient is asymptomatic

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Slide 73: Meeting the Challenge of TB Prevention

For every patient:

  • Assess TB risk factors
  • If risk is present, perform TST or IGRA
  • If TST or IGRA is positive, rule out TB disease
  • If TB disease is ruled out, initiate treatment for LTBI
  • If treatment is initiated, ensure completion

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Slide 74: Additional Resources

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Slide 75: Additional TB Guidelines Available Online

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Slide 76: Case Studies

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Slide 77: Case Study A (1)

Patient History

  • 47-year-old Hispanic male
  • Moved to U.S. from Bolivia 4 years ago
  • Known contact of infectious TB case
  • TST = 5 mm of induration
  • 3 months later TST = 23 mm of induration
  • No symptoms of TB disease
  • Normal CXR, CBC, AST, and bilirubin

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Slide 78: Case Study A (2)

Questions

  • What are this patient’s risk factors for TB infection or disease?
  • Has the management of this patient to date been appropriate?

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Slide 79: Case Study A (3)

Discussion of risk factors

  • Patient is a contact of an infectious TB case
  • Recent  immigrant  to the US from a country with a high prevalence of TB
  • If the patient had not been a contact, the recency of his immigration (less than 5 years) would have made him a candidate for TB testing, but the 5-mm reaction would not be considered positive

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Slide 80: Case Study A (4)

Discussion of risk factors

  • Persons who immigrate from TB-endemic countries have increased rates of TB
  • Rates of TB approach those of their countries of origin for 5 years after arrival in the U.S.
  • These increased rates most likely result from recent M. tuberculosis  infection in their native country

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Slide 81: Case Study A (5)

Discussion of management

  • As a contact of an active TB case, 5 mm of induration is considered positive
  • This patient should have been treated for LTBI immediately after the first TST

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Slide 82: Case Study B (1)

Patient History

  • 24-year-old Asian female
  • Moved to U.S. from Philippines > 5 years ago
  • Plans to work in a correctional facility
  • TST result negative (0 mm) 1 year ago
  • TST for pre-employment physical = 26 mm of induration
  • CXR normal
  • No symptoms of TB disease
  • No known contact with a TB patient

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Slide 83: Case Study B (2)

Questions

  • What are this patient’s risk factors for TB infection or disease?
  • What is the appropriate management for this patient?

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Slide 84: Case Study B (3)

Discussion of risk factors

  • Patient’s TST converted from negative to positive (within a 2-year period)
  • TST conversion increases risk for progressing from LTBI to TB disease
  • Foreign-born status is less of a risk factor, i.e., she immigrated more than 5 years ago

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Slide 85: Case Study B (4)

Discussion of management

  • Patient’s TST conversion indicates failure to identify this person as high risk for recent exposure to TB
  • Patient may have had extended travel to her country of origin or other high-prevalence parts of the world
  • Patient is a recent converter and, as such, is a candidate for treatment of LTBI with INH

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Slide 86: Case Study C (1)

Patient History

  • 28-year-old Asian male
  • Moved to U.S. from China < 5 years ago
  • Received BCG vaccine in China as a child
  • QFT-GIT result = Positive
  • CXR normal
  • No symptoms of TB disease
  • Known contact with a TB patient

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Slide 87: Case Study C (2)

Questions

  • What are this patient’s risk factors for TB infection or disease?
  • What is the appropriate management for this patient?

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Slide 88: Case Study C (3)

Discussion of risk factors

  • Positive QFT-GIT result suggests that M. tuberculosis  infection is likely (result is not affected by prior BCG vaccination)
  • Recent  immigrant  to the US from a country with a high prevalence of TB
  • Foreign-born status is a risk factor, i.e., he immigrated < 5 years ago
  • Known contact with a TB patient

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Slide 89: Case Study C (4)

Discussion of management

  • Patient recently immigrated from a TB endemic country, positive QFT-GIT result may be indicative of LTBI
  • Contact with a TB patient could have been source of infection
  • Should be treated for LTBI

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  17. Q
  18. R
  19. S
  20. T
  21. U
  22. V
  23. W
  24. X
  25. Y
  26. Z
  27. #