231023 P.1.7 GME_PATH_MYCOBACTERIA PART 2.pptx

Mycobacteriology Part 2
Techniques for Identifying Mycobacterium spp.

LEARNING OBJECTIVES
By the end of today’s lecture, you should be able to:
• Describe the different methods used for identifying
Mycobacterium spp.
• Chose the appropriate laboratory methods for diagnosing
mycobacterial infections and mycobacterial disease
• Interpret the results of laboratory identification tests in their
clinical context
• Recognize cases requiring antimicrobial susceptibilities

IDENTIFICATION METHODS
1) Conventional biochemical
2) High performance liquid chromatography (HPLC)
3) Molecular identification techniques
 Direct from specimen (nucleic acid amplification testing)
 From pure cultures (Probe hybridization and sequence-based
techniques)
3) Matrix-assisted laser desorption ionization-time of flight mass spectrometry
(MALDI-TOF MS)
5) Lateral flow assays

BIOCHEMICAL METHODS
• Identification of mycobacteria traditionally relied on phenotypic characteristics
such as:
 Growth rate (rapid vs slow growing)
 Colony morphology and pigmentation (Runyon’s classification)
 Optimal growth temperature
 Biochemicals (e.g. nitrate reduction test, catalase test, tellurite reduction test, etc.)
• Use of biochemicals is no longer recommended for the identification
Mycobacterium spp.
 Many species have not been characterized biochemically
 Variability in phenotypic characteristics exists within strains of a species
 Slow turn around time (weeks)
 Poorly reproducible
Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria. 2018. Clinical Microbiology Reviews; 31(2): 1-66.

HPLC
• Mycobacterial identification based on HPLC analysis of mycolic acid profiles
• Sherlock™ Mycobacterial ID System (MIDI Inc.) is the only commercial platform
available
• HPLC has been replaced in most laboratories by molecular methods for more
accurate species identification (limited library)
Harvesting
Saponification
& Methylation
Extraction
Remove cells from
culture media
Lyse cells to liberate
fatty acids and
methylate
HPLC Analysis & Organism
Identification
Mycolic acid profile is determined and compared
to a library of reference strain profiles
Extract methylated
fatty acid
Modified from: MIDI Inc. Technical Note #101: Bacterial Identification by Gas Chromatographic Analysis of Fatty Acid Methyl Esters (GC-FAME). 2006.

DIRECT SPECIMEN
NUCLEIC ACID AMPLIFICATION TESTING
• Patients with signs and symptoms consistent with pulmonary TB should receive
a TB NAAT on at least one respiratory specimen when possible
 Preference to test first collected specimen to decrease time to result
 AFB positive smears should take priority due to increased sensitivity of NAAT
• NAATs do not replace routine microbiological methods
Two commercial NAATs for
detection of MTBC DNA
directly from respiratory
specimens
Table from: Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria. 2018. Clinical Microbiology Reviews; 31(2): 1-66.

MOLECULAR IDENTIFICATION FROM CULTURES
PROBE HYBRIDIZATION
• Single-stranded DNA probe with a chemiluminescent label binds to the complementary
ribosomal RNA of its target organism and the chemiluminescence from the hybridization can
be detected and quantified
• As no PCR amplification occurs, restricts use of hybridization probes to growth in culture
opposed to direct patient specimen
• Gives result from culture in ~2h
• Culture hybridization probes are highly sensitive and specific - some rare cases of false
positives and negatives:
Polymorphisms preventing hybridization
Cross-reactivity
• Main limitation is that commercial probes are only available
(AccuProbe, Hologic) for: M. tuberculosis complex,
M. intracellulare, M. avium, M. avium complex (MAC),
M. gordonae, and M. kanasii
Caulifield and Wengenack. Diagnosis of active tuberculosis disease: from microscopy to molecular techniques. 2016, J Clin Tuberc Other Mycobact Dis. 4(2016):33-43.

LINE PROBES
• DNA from lysed cultures hybridizes to species-specific probes on a
nitrocellulose membrane
• Hybridization produces colorimetric bands enabling species identification
• Line probes can detect MTBC or common NTM species
• Commercially available line probe assays include:
 INNO-LiPA Mycobacteria assay (Fujirebio)
 GenoType MTBC test (Hain Lifescience)
 Speed-oligo Mycobacteria (Vircell)
Caulifield and Wengenack. Diagnosis of active tuberculosis disease: from microscopy to molecular techniques. 2016, J Clin Tuberc Other Mycobact Dis. 4(2016):33-43.
Sensitivity and specificity: > 90%
Test results within: 4–6 hours

SEQUENCE-BASED TECHNIQUES
• In larger clinical laboratories, sequencing is a primary method to identify
mycobacteria from cultures
• 16s rRNA gene remains the most common target; however, some
mycobacteria are indistinguishable by 16S and require differentiation by other
genes (rpoB, secA, hsp65)
• Some commercially available kits exist for sequencing 16S rRNA (MicroSeq,
ThermoFisher Scientific) but most laboratories use lab-developed protocols
• Based on Clinical and Laboratory Standards Institute (CLSI) guidelines:
 100% identity is needed to report isolate to species level
 99.0-99.9% identity report as “Mycobacterium, most closely related to (species)”
 95.0-98.9% identity report as “Unable to identify by (target) sequencing, most
closely related to Mycobacterium spp.”

• Commonly used method for identification of bacteria from pure cultures
• MALDI-TOF MS detects the abundance of proteins with a specific mass-to-
charge ratio and generates a spectrum (below)
• Spectral data is compared to database to determine
likely identity of the organism
• Cannot differentiate subspecies of M. abscessus
• Two commercially available MALDI-TOF MS:
1) MALDI biotyper (Bruker Daltonics)
2) Vitek MS system (bioMérieux)
MALDI-TOF MS
Image from: Lo et al. AJMR.
2017; 11(35):1360.

LATERAL FLOW ASSAYS
• Contain monoclonal antibody against the MPB64 protein (RD2) secreted by
MTBC
• Reliable, rapid and simple tool to differentiate MTBC members from NTMs in
growth-positive solid or liquid media cultures
• Three commercial tests available:
SD Bioline Ag MPT64 (Alere)
Sensitivity: 97.0%
Specificity: 100%
Capilla TB-Neo (Tauns)
Sensitivity: 98.8%
Specificity: 99.1%
MGIT TBc Identification Test
Sensitivity: 97.9%
Specificity: 99.5%
Brent et al. 2011. J Clin Microbiol; 49: 4343-4346.

SUSCEPTIBILITY TESTING
• Antimicrobial susceptibility testing (AST) should be performed on initial
isolates of M. tuberculosis from all patients and clinically significant isolates
of NTMs
• AST can be repeated 3 months or earlier if a patient remains culture positive
or is not responding to treatment
• Two main methods for AST:
 Phenotypic: growth-based method that tests susceptibility of the organism to a
critical drug concentration
 Molecular: detects mutations in genes associated with resistance
• Molecular methods are rapid and specific, but phenotypic susceptibility must
still be performed as genotypic resistance may not always be translatable to
phenotypic resistance

COMMONLY USED PHENOTYPIC AST METHODS FOR
MTBC
**Broth microdilution assay is the gold-standard method for NTMs; however, the role and relevance of in vitro AST of
NTM to guide treatment is under continuous debate. Unlike M. tuberculosis, the clinical response of NTMs to
antimycobacterial drugs has been shown to correlate with only some compounds and in only some NTMs.

COMMONLY USED MOLECULAR AST METHODS FOR
MTBC

APPROACH TO DIAGNOSIS TUBERCULOSIS INFECTION
(LTBI)
Approach to diagnosis Tuberculosis infection (LTBI) − Identification and
treatment of individuals with latent tuberculosis (TB) infection (LTBI) is an
important priority for TB control. LTBI is a clinical diagnosis that is established by
demonstrating prior TB infection and excluding active TB disease. The approach
to diagnosis of LTBI is discussed separately.
Available assays:
• Tuberculin skin test (TST)
• Interferon-gamma release assays (IGRAs).
2023 UpToDate

USE OF THE TST TEST FOR DIAGNOSIS OF TUBERCULOSIS
INFECTION (TUBERCULOSIS SCREENING) IN ADULTS
Tools for diagnosis of TB infection – TB infection is a clinical diagnosis that is established by
demonstrating prior TB infection and excluding TB disease. Available tests to demonstrate prior
TB infection include the tuberculin skin test (TST) and interferon-gamma release assays
(IGRAs). Issues related to use of IGRAs are discussed separately.
Interpreting the TST – The TST assesses the intradermal delayed-type hypersensitivity
response produced by administration of purified protein derivative (PPD) from Mycobacterium
tuberculosis.
Negative tests
• False-negative tests – TST can be falsely negative because of biologic problems (such as
immunosuppression) or because of technical problems with the TST administration, PPD
material, or reading. The TST result can be false positive because of infection with
nontuberculous mycobacteria or prior immunization with Bacillus Calmette-Guérin (BCG).
• Management of individuals with negative test – For immunosuppressed individuals who
have had recent close contact with known infectious cases of TB, treatment for TB infection
may be started even if the initial TST is negative. In such cases, testing for TB infection
should be repeated after eight weeks. If the second test is positive, treatment is continued; if
the second test is negative, treatment is usually stopped.
2023 Up ToDate

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Measuring a reaction to the tuberculin skin test (TST)
Bacille Calmette-Guérin vaccination scarification

Positive tests – Patients with positive TST or IGRA results must undergo
clinical evaluation to rule out TB disease and to assess need for treatment of TB
infection. This includes evaluation for symptoms (eg, fever, cough, weight loss),
physical exam, and radiographic examination of the chest.
Repeat testing – A period of up to eight weeks after primary TB infection may
be required for TST conversion to occur. Individuals with close contact to a
known infectious case of TB and negative initial TST should have repeat TST 8
to 10 weeks after the end of exposure.
Serial testing – Serial testing is warranted for individuals with ongoing potential
exposure. This includes residents or employees of homeless shelters or
correctional facilities, and health care workers in some circumstances. In these
settings, TST is the test of choice, given issues of false conversions and
reproducibility with IGRAs. Those undergoing serial annual testing should also
undergo testing after a known episode of exposure.
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INTERFERON-GAMMA RELEASE ASSAYS (IGRA)
IGRAs are diagnostic tools for LTBI. They are in vitro blood tests of cell-mediated
immune response to Mycobacterium tuberculosis and measure T cell release of
interferon-gamma following stimulation by antigens specific to M. tuberculosis.
IGRAs cannot distinguish between latent infection and active TB disease and
should not be used for diagnosis of active TB. A negative IGRA does not rule out
active TB at any age.
Types of IGRA assays:
• QuantiFERON-TB Gold In-Tube Plus (QFT-GIT Plus) assay
• T-SPOT.TB assay is an enzyme-linked immunospot performed on separated
peripheral blood mononuclear cells (PBMCs)
• QFT-GIT Plus is an enzyme-linked immunosorbent assay-based whole blood
test.
2023 UpToDate

INTERFERON-GAMMA RELEASE ASSAYS (IGRA).
Sensitivity and specificity
• The sensitivity for T-SPOT.TB appears to be higher than QFT-GIT or TST, likely because the
testing platform ensures that an adequate number of PBMCs are available even in the
presence of low lymphocyte cell counts in whole blood.
• The sensitivity and specificity of the QFT-Plus assay appears to be comparable with the
preceding versions of the QuantiFERON test. However, there is insufficient evidence of the
diagnostic accuracy of this test in patients with HIV infection or children.
IGRAs preferred for patients with BCG vaccination − IGRAs are not affected by Bacille
Calmette-Guérin (BCG) vaccination status and have specificity >95 percent for diagnosis of
LTBI. Therefore, IGRAs are preferred for patients with history of BCG vaccination, especially if
received in countries where BCG is given following infancy or are repeated.
IGRAs should not be used for serial testing − For serial testing in populations with ongoing
TB exposure, data are insufficient for interpretation of IGRA conversions and reversions, and
most studies suggest poor assay reproducibility. In these settings, performance of the TST by
specifically trained personnel is the test of choice.
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A COMPARISON OF TUBERCULIN SKIN TESTS AND
INTERFERON-GAMMA RELEASE ASSAYS
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CASE 1
• 44 year old (HIV negative) patient was recently diagnosed with relapsed
pulmonary TB, and her isolate was reported as susceptible to first line
therapy
• After a month of therapy, she is doing well but develops new pulmonary
infiltrates
• Sputum AFB smears are negative but want to make sure her current
treatment regimen is working and request NAAT (PCR) on the specimen
• The laboratory cancels your request for NAAT testing so you collect another
sputum specimen and send to an outside laboratory that uses the Cepheid
Xpert system
• Her sputum is positive for M. tuberculosis and detects rifampicin resistance

NEXT STEPS
1. Why did the laboratory cancel the request for NAAT on sputum?
1Boyles et al. Int J Tubercul Lung Dise. 2014. 18(7): 876-878; 2Ocheretina et al. Diagn Microbiol Infect Dis. 2016. 85(1): 53-55;3Hanrahan et al. Am J Respir Crit Care Med. 2014. 189(11): 1426-1434.
• Conversion of Xpert to negative is not a suitable marker for treatment
success as it cannot distinguish dead from living bacilli
• ~30% of patients remain Xpert positive after 6 months of therapy, although
only 4% are positive by culture1
• Xpert results having a “very low” M. tuberculosis detection level were reported
to have a ~64% false positive rate for rifampin resistance2
• Since 86% of cases AFB smear-negative samples test “very low” with Xpert3, it
is likely that the result is a false positive
2. How do you interpret the Xpert rifampin resistant result?

NEXT STEPS
4. Would you request additional susceptibility testing?
• Xpert rifampin-resistant diagnoses in tests with “very low” M. tuberculosis detection
should be confirmed with culture-based susceptibility testing
• Repeat susceptibility testing is generally recommended if cultures fail to convert to
negative after 3 months or if there is clinical evidence of failure to respond to
therapy1
• Inflammatory response associated with TB treatment (paradoxical reaction)
occurs in 10% of patients2
• TB itself is immunomodulatory, and initiation of treatment reverses immune
dysregulation
5. No growth was observed on repeat sputum cultures suggesting
successful therapy. What could be the cause of her new pulmonary
infiltrates? What additional information would you want to know?
1Clinical and Laboratory Standards Institute. M24-A2: Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; 2Breen et al. Thorax. 2004. 59:704-707.

CASE 2
• 9 year old male is immigrating to the United States with his parents from a
TB endemic country
• He undergoes a physical examination as part of his immigration medical
screening which indicates he is otherwise healthy, but his interferon-gamma
release assay (IGRA) result is positive
• A chest x-ray demonstrates radiographic evidence of TB
• The child must have 3 sputum smears and cultures for M. tuberculosis prior
to immigration
Centres for Disease Control and Prevention. Guidelines for Screening for Tuberculosis Infection and Disease during the Domestic Medical Examination for Newly Arrived Refugees

NEXT STEPS
1. What are the challenges surrounding laboratory diagnosis of pediatric
TB?
• Difficult to collect sputum specimens
• Sputum smears and culture are less sensitive due to lower bacterial loads in
children
• An early morning gastric aspirate before the child arises and peristalsis
empties the stomach of the respiratory secretions swallowed overnight
2. The clinical team was not able to collect sputum from the child. What
is the optimal specimen for pediatric pulmonary TB?

NEXT STEPS
3. All gastric aspirate smears were negative. Would you expect these
results in this patient?
• Yes, gastric aspirates smears also have poor sensitivity
• Three consecutive morning gastric aspirates yield M. tuberculosis in only 30-
50% of active pulmonary TB cases1
• Xpert Mtb/RIF can be used to diagnosis pulmonary TB in children
 66% sensitivity and >98% specificity2
4. The family asks you if there are any other tests that can be used to
diagnosis their son sooner? What do you suggest?
1Vallejo et al. 1994. Pediatrics. 94:1-7; 2 WHO. 2013. The use of Xpert MTB/RIF assay for the detection of pulmonary and extrapulmonary TB and rifampicin resistance in adults and children.

NEXT STEPS
5. Xpert MTB/RIF results are negative on all three gastric aspirates;
however, after 3 weeks of incubation growth is observed from all three
specimens.
What steps does the laboratory take to identify the organism?
Colonies from patient
gastric aspirates
Figure from: Association of Public Health Laboratories https://www.aphl.org/programs/infectious_disease/tuberculosis/TBCore/Identification_of_Mycobacteria_FINAL.pdf

NEXT STEPS
however, after 3 weeks of incubation at 37°C, growth is observed from
all three specimens.
What steps does the laboratory take to identify the organism?
(i) Confirm acid fastness of the organism
Kinyoun stain: photo courtesy of the Hospital of the University of Pennsylvania; Table modified from Koh. Nontuberculous Mycobacteria – Overview. 2017; Microbiology Spectrum. 5(1):1-7
Kinyoun stain from culture plate (100x)
(ii) Based on growth rate and pigmentation
what organism(s) do you suspect?

NEXT STEPS
however, after 3 weeks of incubation at 37°C, growth is observed from
all three specimens.
(iii) What tests could the laboratory perform to identify the organism?
1. HPLC
2. Probe Hybridization
3. Line Probes
4. MALDI-TOF MS
5. 16s rRNA Sequencing

NEXT STEPS
6. The MALDI-TOF MS identifies the organism as Mycobacterium
gordonae. The isolate is sent out to a reference laboratory and 16s rRNA
sequencing confirms the identification.
Based on these results, how would you manage the patient? What
treatment does he require?
• M. gordonae is rarely clinically
significant, even in
immunocompromised patients
• Considered a contaminant in
laboratory cultures
• Patient should be treated for latent
TB
Figure from: Association of Public Health Laboratories https://www.aphl.org/programs/infectious_disease/tuberculosis/TBCore/Identification_of_Mycobacteria_FINAL.pdf

CASE 3
• 49 year old woman presents with a 2-month history of multiple painful
nodules and abscesses on her forehead and periorbital areas following botox
treatment
• Lesions initially started as erythematous papules and nodules on all of the
injection sites 1 week after botox injections and gradually developed
abscesses with ulceration and crusting
• You suspect an allergic reaction with secondary impetiginisation so you treat
her with prednisone and azithromycin
• Lesions recurred 1 week after prednisone was discontinued
• A skin biopsy was performed

NEXT STEPS
1. Histopathology identifies polymorphonuclear microabscesses and
epithelioid granuloma formation. Based on these results you request a
FITE stain. Is the FITE stain positive or negative for AFB?
AFB
Photo courtesy of the Hospital of the University of Pennsylvania
Skin biopsy FITE stain (40x)
Skin biopsy reveals
3+ AFB

NEXT STEPS
Within 5 days of incubation at 37°C, the solid culture media
shows mature colonies.
ITable modified from Koh. Nontuberculous Mycobacteria – Overview. 2017; Microbiology Spectrum. 5(1):1-7;
Table modified from Koh. Nontuberculous Mycobacteria – Overview. 2017; Microbiology Spectrum. 5(1):1-7;
Image from: http://atlas.actino.jp/data/1/1-57.jpg Kinyoun stain of culture (100x)
Photo courtesy of the Hospital of the University of Pennsylvania

NEXT STEPS
2. What species do you suspect based on growth rate?
Tables from Koh. Nontuberculous Mycobacteria – Overview. 2017; Microbiology Spectrum. 5(1):1-7.

NEXT STEPS
3. What methods can the laboratory use to identify the organism?
1. HPLC
2. Line Probes
3. MALDI-TOF MS
4. Gene sequencing
4. The laboratory identifies the organism as M. abscessus complex by
MALDI-TOF MS. Should the laboratory perform additional testing?
• M. abscessus complex is further divided into 3 subspecies:
 M. abscessus subsp. abscessus
 M. abscessus subsp. massiliense
 M. abscessus subsp. bolletii
• Subspecies differ in susceptibility profiles due to the presence of erm(41) gene
• MALDI-TOF MS cannot differentiate subspecies, rpoB gene sequencing required

MACROLIDE RESISTANCE IN M. ABSCESSUS COMPLEX
• erm(41) gene in M. abscessus complex confers macrolide resistance through
methylation of 23S ribosomal RNA
• Majority of strains of M. abscessus subsp. massiliense have a
truncated erm(41) gene rendering the organism susceptible to macrolides
• Some subspecies can have inducible resistance to clarithromycin, where that
can be observed in vitro following prolonged incubation (14 days)
Zhu et al. 2015. Mol Cell Probes. 29(6):514-516.

TAKE HOME POINTS
• Mycobacterial species cannot be correctly identified based on phenotypic
characteristics alone – rely on molecular techniques
• Biochemicals are rarely used for the identification of mycobacteria due to
slow turn around time and poor specificity
• Current NAAT techniques (e.g. Xpert MTB/RIF) on direct specimens are
limited to MTBC, nontuberculous mycobacteria require growth from culture
• MALDI-TOF MS can differentiate species of the MTBC and MAC but cannot
different subspecies of M. abscessus

TAKE HOME POINTS
• Certain mycobacteria are indistinguishable by 16s rRNA sequencing but can
be differentiated by sequencing other genes such as rpoB, secA, or hsp65
• Since species are so closely related, 100% sequence identity to a database
is needed to identify to the species level
• Molecular techniques cannot be used to test for cure
• REMEMBER: not all mycobacteria represent pathogens. Before initiating
treatment:
 Link clinical symptoms with microbiological findings
 Ensure appropriate exclusion of other diagnoses

SUGGESTED READINGS
Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories:
Mycobacteria. 2018. Clinical Microbiology Reviews; 31(2): 1-66.
Caulifield and Wengenack. Diagnosis of active tuberculosis disease: from
microscopy to molecular techniques. 2016, J Clin Tuberc Other Mycobact
Dis. 4(2016):33-43
World Health Organization. Using the Xpert MTB/RIF assay to detect
pulmonary and extrapulmonary tuberculosis and rifampicin resistance in
adults and children. 2013. https://www.who.int/tb/publications/xpert-mtb-
rif-assay-diagnosis-meeting-report/en/

231023 P.1.7 GME_PATH_MYCOBACTERIA PART 2.pptx

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