Mycobacteria

19 DOH Abu Dhabi (HAAD) lab licensing practice questions on Mycobacteria, part of Microbiology. The first 19 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   A 29-year-old woman lives with a relative who was treated for TB in another country; his treatment outcome is unknown. She has had cough and night sweats for 6 weeks. Sputum results: Ziehl-Neelsen smear 2+; Xpert MTB/RIF 'MTB detected medium; RIF resistance not detected'. The MGIT culture flags positive on day 11, the broth shows corded acid-fast bacilli, and the MPT64 antigen test on the broth is positive. Before the isolate is reported as drug-susceptible TB, which further test is most important?

   1. AA second Xpert MTB/RIF on the cultured isolate
   2. BIsoniazid susceptibility by line probe assay or DST
   3. CFluoroquinolone and linezolid susceptibility only
   4. DNo further test; Xpert has excluded resistance

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   Answer: B · Isoniazid susceptibility by line probe assay or DST

   The corded, MPT64-positive isolate confirms M. tuberculosis complex. Xpert MTB/RIF examines only the rpoB core region, so it predicts rifampicin resistance but gives no information on isoniazid. Isoniazid-resistant, rifampicin-susceptible TB (Hr-TB) is common worldwide and is missed when Xpert is the only test, and a contact of a patient with an unknown outcome is at higher risk of resistance. WHO recommends testing for isoniazid resistance, by a line probe assay that detects katG and inhA mutations, a newer molecular assay or phenotypic DST, so the regimen can be changed if needed. Second-line testing for fluoroquinolones is prioritised when rifampicin resistance is detected.

   • A: Repeating Xpert retests the same rpoB region and adds nothing on isoniazid.
   • C: Second-line testing is the priority when rifampicin resistance is found.
   • D: Xpert MTB/RIF detects rifampicin resistance only, not isoniazid resistance.

2. Question 2 · Hard

   A 41-year-old man applying for a UAE residence visa has a nodular opacity in the right upper lobe on his chest radiograph and has never been treated for TB. Three sputum samples are sent. Results: auramine smear positive (1+) on two samples, confirmed by Ziehl-Neelsen stain; Xpert MTB/RIF Ultra on a smear-positive sample 'MTB not detected', with a valid internal control; MGIT culture positive on day 9, with acid-fast bacilli in the broth that do not form cords; MPT64 immunochromatographic test on the broth negative. The most likely interpretation is:

   1. ASmear-positive TB missed by Xpert; treat as TB
   2. BM. tuberculosis lacking MPT64; repeat the MPT64 test
   3. CLaboratory cross-contamination; report as negative
   4. DA nontuberculous mycobacterium; identify to species

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   Answer: D · A nontuberculous mycobacterium; identify to species

   Acid-fast bacilli are seen in two of the patient's own sputum smears and in the MGIT broth, so a mycobacterium is present. Xpert MTB/RIF Ultra detects M. tuberculosis complex in almost all smear-positive samples, so 'not detected' with a valid internal control argues strongly against TB. The negative MPT64 test and the absence of cording point the same way, to a nontuberculous mycobacterium. The isolate should be identified to species by line probe assay, MALDI-TOF or sequencing, because the significance and treatment of M. avium complex, M. kansasii and M. abscessus differ. Cross-contamination cannot explain bacilli in the patient's direct smears.

   • A: Xpert Ultra detects nearly all smear-positive TB, so a negative result argues against TB.
   • B: Rare MTBC strains lack MPT64, but Xpert would still detect them.
   • C: The bacilli were seen in the patient's own direct smears, so cross-contamination does not explain them.

3. Question 3 · Hard

   A 58-year-old former miner has a cavitary upper-lobe lesion. Sputum grows acid-fast bacilli in 2 weeks at 37 °C. Colonies are buff in the dark and turn yellow after light exposure. The isolate is nitrate positive, Tween 80 hydrolysis positive and strongly 68 °C catalase positive. The most likely organism is:

   1. AMycobacterium kansasii
   2. BMycobacterium tuberculosis
   3. CMycobacterium gordonae
   4. DMycobacterium marinum

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   Answer: A · Mycobacterium kansasii

   M. kansasii is a slowly growing photochromogen: colonies grown in the dark are buff and turn yellow after exposure to light as carotene forms. It is nitrate positive, hydrolyses Tween 80 and has strong heat-stable (68 °C) catalase, unlike M. tuberculosis. It causes TB-like cavitary lung disease, often in older men with damaged lungs such as from pneumoconiosis. Most laboratories now confirm such phenotypic results by molecular methods or MALDI-TOF.

   • B: M. tuberculosis is nonpigmented and negative for 68 °C catalase.
   • C: M. gordonae is a scotochromogen, pigmented even in the dark, and nitrate negative.
   • D: M. marinum is also a photochromogen but is nitrate negative and grows poorly at 37 °C.

4. Question 4 · Hard

   A TB laboratory's culture contamination rate rises to 12%, against a target of 2% to 5%. Most affected specimens came from outlying clinics and arrived 3 to 4 days after collection without refrigeration. What is the most likely cause?

   1. ACentrifugation speed set too low
   2. BIncubation of solid media without CO2
   3. COvergrowth of flora during delayed, unrefrigerated transport
   4. DNaOH concentration set too high in the digestant

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   Answer: C · Overgrowth of flora during delayed, unrefrigerated transport

   Normal respiratory flora multiply in sputum held at room temperature, and a heavier load is more likely to survive decontamination and overgrow the culture. Specimens that cannot be processed within about an hour should be refrigerated and transported as quickly as possible. Too much NaOH would lower contamination, while killing mycobacteria too. Low centrifugal force reduces recovery of mycobacteria, and lack of CO2 slows their growth; neither raises the contamination rate.

   • A: Low centrifugal force reduces recovery, not contamination.
   • B: Lack of CO2 slows mycobacterial growth but does not add contaminants.
   • D: Excess NaOH lowers contamination and kills mycobacteria too.

5. Question 5 · Medium

   Why is a decontaminated sputum specimen centrifuged at 3,000 × g or more for 15 to 20 minutes before mycobacterial culture?

   1. AIt pellets the mucus while the bacilli stay in solution
   2. BLipid-rich mycobacteria are buoyant and sediment poorly
   3. CThe force breaks up cords so colonies can be counted
   4. DIt kills the normal flora that survive the NaOH

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   Answer: B · Lipid-rich mycobacteria are buoyant and sediment poorly

   The waxy, lipid-rich cell wall makes mycobacteria buoyant, so they sediment slowly. A relative centrifugal force of at least 3,000 × g for 15 to 20 minutes concentrates them in the pellet, which is used for the smear and the cultures. Lower forces lose organisms and reduce the sensitivity of both smear and culture. Centrifugation is done in sealed safety cups that are opened only inside a biological safety cabinet.

   • A: The bacilli are wanted in the pellet; the supernatant is discarded.
   • C: Cords are not broken up for counting; the pellet is cultured directly.
   • D: Centrifugation does not kill bacteria; NaOH does the decontamination.

6. Question 6 · Hard

   A new patient with no previous TB treatment and no contact with drug-resistant TB has sputum Xpert MTB/RIF 'MTB detected high; RIF resistance detected'. Local rifampicin resistance is under 2%. What is the best next laboratory step?

   1. AConfirm on a second sample and send for DST
   2. BRequest an IGRA to confirm the TB
   3. CReport the isolate as XDR-TB
   4. DDismiss the resistance result as an artefact

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7. Question 7 · Hard

   A MGIT tube from sputum flags positive on day 6. A Ziehl-Neelsen smear of the broth shows no acid-fast bacilli but many blue-stained cocci, and a blood agar subculture grows heavily overnight. What is the best interpretation and action?

   1. AM. tuberculosis growth; report the culture as positive
   2. BBacterial contamination; decontaminate again and re-culture
   3. CRapidly growing mycobacteria; start identification
   4. DInstrument false signal; return the tube until day 42

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8. Question 8 · Medium

   Xpert MTB/RIF Ultra on a sputum sample reports 'MTB detected trace; RIF resistance indeterminate'. Why is no rifampicin result given?

   1. AThe organism is a nontuberculous mycobacterium
   2. BToo little DNA to assess the rpoB region
   3. CRifampicin resistance cannot be tested on sputum
   4. DThe sample contained an amplification inhibitor

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9. Question 9 · Hard

   A visa-screening applicant has old fibrotic scarring on his chest radiograph and completed TB treatment 2 years ago. Sputum results: AFB smear negative; Xpert MTB/RIF Ultra 'MTB detected trace'; MGIT culture no growth at 42 days, with valid controls. What is the most likely explanation?

   1. AActive rifampicin-resistant tuberculosis
   2. BAmplicon carryover from an earlier cartridge run
   3. CDNA from nonviable bacilli of past disease
   4. DCross-reaction with a nontuberculous mycobacterium

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10. Question 10 · Medium

    A visa-screening applicant with an abnormal chest radiograph has a negative sputum AFB smear, so culture and NAAT are requested. A smear usually becomes positive only when sputum contains about:

    1. A1 million or more bacilli per mL
    2. B5,000 to 10,000 bacilli per mL
    3. C100 to 200 bacilli per mL
    4. D10 to 100 bacilli per mL

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11. Question 11 · Medium

    Which urine specimen is best for mycobacterial culture in suspected renal tuberculosis?

    1. AA 24-hour pooled urine collection
    2. BFirst-morning urine on 3 consecutive days
    3. CA single random catheter urine
    4. DMidstream urine collected after a diuretic

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12. Question 12 · Medium

    A positive MGIT broth contains acid-fast bacilli. An immunochromatographic test on the broth detects MPT64 antigen. This identifies the isolate as:

    1. AM. kansasii
    2. BM. tuberculosis complex
    3. CM. abscessus
    4. DM. avium complex

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13. Question 13 · Hard

    A molecular test on a sputum isolate shows an rpoB mutation and a katG mutation. A second-line line probe assay then detects a gyrA mutation. Linezolid and bedaquiline results are not yet available. By WHO 2021 definitions, how is the isolate classified so far?

    1. APre-extensively drug-resistant TB (pre-XDR)
    2. BExtensively drug-resistant TB (XDR)
    3. CMultidrug-resistant TB (MDR) only
    4. DIsoniazid-resistant, rifampicin-susceptible TB

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14. Question 14 · Hard

    A smear-negative sputum from a patient whose pneumonia resolved on routine antibiotics grows one colony of M. tuberculosis complex after 5 weeks. It was processed in the same batch, right after a 3+ smear-positive specimen. What is the best next step?

    1. ARepeat the identification with an MPT64 test
    2. BReport TB and advise starting four-drug therapy
    3. CGenotype both isolates to check for cross-contamination
    4. DDiscard the colony as a nontuberculous contaminant

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15. Question 15 · Easy

    Before a mycobacterial culture is reported as negative, it is usually incubated for:

    1. A5 days
    2. B6 weeks
    3. C6 months
    4. D2 weeks

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16. Question 16 · Medium

    Early-morning gastric aspirates are collected from a 4-year-old with suspected pulmonary TB. They cannot be processed until the next day. What should be done to them?

    1. AFreeze them at -20 °C until processing
    2. BNeutralise the stomach acid without delay
    3. CAdd NaOH and leave them overnight
    4. DFix them in formalin to keep the bacilli

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17. Question 17 · Easy

    In NALC-NaOH processing of sputum for mycobacteria, the sodium hydroxide serves to:

    1. AKill contaminating bacteria
    2. BLiquefy the mucus
    3. CNeutralise the specimen
    4. DStain the acid-fast bacilli

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18. Question 18 · Easy

    Middlebrook 7H10 and 7H11 agar for mycobacterial culture are incubated at:

    1. A35 to 37 °C in 5% to 10% CO2
    2. B42 °C in ambient air
    3. C30 °C under anaerobic conditions
    4. D25 °C in ambient air

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19. Question 19 · Medium

    Besides Mycobacterium tuberculosis DNA, what does the Xpert MTB/RIF assay detect?

    1. AHIV RNA
    2. BRifampicin resistance mutations in rpoB
    3. CIsoniazid resistance mutations in katG
    4. DEthambutol resistance

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