LABORATORY METHODS: TUBERCULOSIS DIAGNOSIS...Curry International Tuberculosis Center May 8-11, 2018 Laboratory methods: tuberculosis diagnosis TB Case Management and Contact Investigation

TB CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE May 8-11, 2018

Curry International Tuberculosis Center, UCSF

300 Frank H. Ogawa Plaza, Suite 520

Oakland, CA; Office (510) 238-5100

LABORATORY METHODS:

TUBERCULOSIS DIAGNOSIS

LEARNING OBJECTIVES

Upon completion of this session, participants will be able to:

1. Describe three laboratory methods used in the diagnosis and control of TB resulting in a better

understanding of laboratory results and improved communication between the clinician, the

laboratory, and the patient

INDEX OF MATERIALS PAGES

1. Laboratory Methods: Tuberculosis Diagnosis – slide outline Presented by: Ed Desmond, Ph.D.

1-27

SUPPLEMENTAL MATERIAL

None





ADDITIONAL REFERENCES

• CDC. Availability of an Assay for Detecting Mycobacterium tuberculosis, Including Rifampin- Resistant Strains, and Considerations for Its Use – United States, 2013. MMWR 2013;62 (No.41) October 18, 2013.

• CDC. Guide to the application of genotyping to tuberculosis prevention and control.

Page last updated September 1, 2012. www.cdc.gov/tb/programs/genotyping/manual.htm

• CDC. Report of an expert consultation on the uses of nucleic acid amplification tests for the diagnosis of tuberculosis. Page last updated September 1, 2012. www.cdc.gov/tb/publications/guidelines/amplification_tests/background.htm

• WHO. Drug-resistant tuberculosis. Frequently asked questions. January 2012. http://www.who.int/tb/challenges/mdr/tdrfaqs/en/# (Accessed November 10, 2014).

• CDC. Updated Guidelines for Using Interferon Gamma Release Assays to Detect

Mycobacterium tuberculosis Infection, United States. MMWR 2010; 59 (No.RR-5) See CDC factsheet Interferon-Gamma Release Assays (IGRAs). Page last updated September 1, 2012. http://www.cdc.gov/tb/publications/factsheets/testing/IGRA.htm

• Chang-Hong, S., Xiao-Wu, W., Hai, Z., et al. Immune responses and protective efficacy

of the gene vaccine expressing Ag85B and ESAT6 fusion protein from Mycobacterium tuberculosis. DNA Cell Biol. 2008 Apr;27(4):199-207.

• Honscha, G., Von Groll, A., Valenca, M., et al. The laboratory as a tool to qualify

tuberculosis diagnosis. Int. J. Tuberc Lung Dis. 2008;12(2):218-20.

• Perez-Martinez, I., Ponce-De-Leon, A., Bobadilla, M., et al. A novel identification scheme for genus Mycobacterium, M.tuberculosis complex, and seven mycobacteria species of human clinical impact. Eur. J. Clin. Microbiol. Infect. Dis. 2008;27(6):451-459.

• Barman, P., Gadre, D., A study of phage based diagnostic technique for tuberculosis. Indian J. Tuberc. Jan. 2007; 54(1):36-40.

• Haldar, S., Chakravorty, S., Bhalla, M., De Majumdar, S., Tyagi, JS. Simplified detetion of Mycobacterium tuberculosis in sputum using smear microscopy and PCR with molecular beacons. J. Med. Microbiol. 2007;56(Pt.10):1356-62.

• Nahid, P., Pai, M., Hopewell, P. Advances in the diagnosis and treatment of tuberculosis. Proc. Am. Thorac. Soc. 2006; 3:103-110.

http://www.cdc.gov/tb/programs/genotyping/manual.htm

http://www.cdc.gov/tb/publications/guidelines/amplification_tests/background.htm

http://www.who.int/tb/challenges/mdr/tdrfaqs/en/

http://www.cdc.gov/tb/publications/factsheets/testing/IGRA.htm





• Somoskovi, A., Dormandy, J., Mitsani, D., Rivenburg, J., Salfinger, M. Use of smearpositive samples to assess the PCR-based genotype MTBDR assay for rapid, direct detection of the Mycobacterium tubercuslosis complex as well as its resistance to isoniazid and rifampin. J. Clin. Microbiol. 2006;44(12):4459-4463.

• Lin, G., Probert, W., Lo, M., Desmond, E. Rapid detection of isoniazid and rifampin

resistance mutations in Mycobacterium tuberculosis complex from cultures or smearpositive sputa by use of molecular beacons. J. Cli. Microbiol. 2004;42(5):4204-4208.

• Barnes, P., Cave, D. Molecular epidemiology of tuberculosis. NEJM. 2003;349(12): 1149-1155. Review article.

• Dowdy, D.W., Maters, A., Parrish, N., Beyer, C., Dorman, S.E. Cost-effectiveness

analysis of the gen-probe amplified mycobacterium tuberculosis direct test as used routinely on smear-positive respiratory specimens. J. Clin. Microbiol. 2003;41(3):948-53.

• Drobniewski, F.A., Caws, M., Gibson, A., Young, D. Modern laboratory diagnosis of

tuberculosis. Lancet Infect. Dis. 2003;3(3):141-47.

• Van Der Zanden, A.G., Te Kopppele-Vije, E.M., Vijaya Bhanu, N., et al. Use of DNA extracts from Ziehl-Neelsen-stained slides for molecular detection of rifampin resistance and spoligotyping of Mycobacterium tuberculosis. J. Clin. Microbiol. 2003;41(3):1101-08.

• Gillespie, S. Minireview. Evolution of drug resistance in Mycobacterium tuberculosis:

clinical and molecular perspective. Antimicrob. Agents Chemother. 2002;46(2):267-274.

• Mostowy, S., Behr, M.A., Comparative genomics in the flight against tuberculosis: diagnostics, epidemiology and BCG vaccination. Am. J. Pharmacogenomics. 2002;2(3):189-196.

• Somoskovi, A., Mester, J., Hale, Y.M., Parsons, L.M., Salfinger, M. Laboratory diagnosis of nontuberculous mycobacteria. Clin. Chest Med. 2002;23(3):585-597.

• Breese, P.E., Burman, W.J., Hildred, M., et al. The effect of changes in laboratory

practices on the rate of false-positive cultures for Mycobacterium tuberculosis. Arch Pathol. Lab Med. 2001;125(9):1213-1216.

• Somoskovi, A., Parsons, L.M., Salfinger, M. The molecular basis of resistance to

isoniazid, rifampin, and pyrazinamide in Mycobacterium tuberculosis. Respir. Res. 2001;2(3):164-168.

• Hale, Y.M., Desmond, E.P., Jost, K.C., Jr., Salfinger, M. Access to newer laboratory procedures: a call for action. Int. J. Tuberc Lung Dis. 2000;4(12 suppl 2):S171-175.

Curry International Tuberculosis CenterMay 8-11, 2018

Laboratory methods: tuberculosis diagnosis

TB Case Management and Contact Investigation Intensive

1

LABORATORY

METHODS:

Tuberculosis Diagnosis

Ed Desmond

Microbial Diseases Lab, Calif. Dept. of Public Health

Richmond, CA (510) 412-3781

[email protected]

Specimen collection and transport

Specimens (sputum, bronchial washings, urine,

etc.) should be collected in a laboratory-approved

sterile, leak-proof, non-breakable container

Containers must be labeled with patient’s name

and date collected

Begin collecting specimens prior to initiation of

therapy




2

Collection and transport (2)

Sputum is the most common specimen

• Collect 5-10 mls of an early morning

specimen, prior to eating

• Usually 3 specimens on 3 different days

are recommended for diagnosis


Contaminated specimens can be minimized by

• Instructing the patient to rinse mouth with

preferably sterile water before collecting the

specimen

• Returning the specimen to the lab as soon

as feasible after collection




3


Indicate type of specimen on laboratory

requisition form

Keep all specimens refrigerated and transport

as soon as possible to the lab

How many specimens to collect?

The greater the number of specimens, the higher the

probability of a positive

Law of diminishing returns: 4 specimens doesn’t give

many more positives than 3, so 3 is usual guideline




4

Sputum smear microscopy resultsaccording to the specimen collection

Study Specimen

Type

Total % Positive

1 Overnight 160 85,0

Spot 160 51,8

2 Overnight 181 31,5

Spot 179 13,9

1 PANDE et al., Indian J Tuberc 21:1974, 192

2 R.VALLADERS & R.URBANCZIK, Instituto Nacional de TB, Venezuela

1968 – 1969 (not published)

POSITIVITY ON SMEAR RELATED TO QUALITY OF SPUTUM SPECIMENS

Group No. of speci-

mens in each

group

Per cent of

specimens

Number

positive

Per cent

positive

Saliva 1125 32.9 21 1.8

Mucous 1707 49.9 68 3.5

Mucopu-

rulent

583 17.2 220 37.7

Note that muco purulent specim ens are positive more often than saliva or mucous.

L.POLLAK & R.URBANCZIK, Bol Inform Inst Nac Tuberc (Caracas) 2:1969,5 - 8




5

Processing pulmonary specimens

Digestion and decontamination

• Pulmonary specimens are exposed to a

mucolytic agent to dissolve mucin and

liquefy the specimen

• N-acetyl-L-cysteine (NALC) is the most

common mucolytic agent used

Processing pulmonary specimens (2)

Digestion and decontamination

• Specimens are also treated with a liquid

decontaminant, generally sodium

hydroxide, a strong alkali which is more

toxic to oral flora than AFB

• Material is concentrated by centrifugation




6

Staining concentrated smear

Fluorochrome stains

• Fluorochrome stained smears require a

fluorescent microscope

• Generally read at 250X-450X magnification

which allows rapid scanning of the smear

• Auramine-rhodamine is an example of such a

stain where the AFB appear yellow against a

black background




7

Carbol fuchsin-based stains

• Utilize a regular light microscope

• Must be read at a higher magnification

• Two types: Ziehl-Neelsen and Kinyoun.

Both use carbol fuchsin/phenol as the

primary dye (NOTE: ZN is more sensitive)

• Smear is then decolorized with acid-(HCI)

alcohol and counter-stained with

methylene blue

Staining concentrated smear (2)

From CDC Lab Manual




8

REPORTING AFB SMEAR RESULTS*

Number of AFB found: Report:

0 — or Neg

1-2 / 300 fields

1-9 / 100 fields 1+

1-9 / 10 fields 2+

1-9 / field 3+

>9 / field 4+

*CDC System (WHO system goes up to 3+ only)

Inoculating growth media for culture

Solid media

Two types most commonly used are

• Lowenstein-Jensen which is egg-based

• Middlebrook 7H 10 or 7H11 which are agar-

based




9

Inoculating growth media for culture (2)

Solid media have the advantage that organisms (colonies) can be seen on the surface of the medium

If there is mixed growth or contamination, picking individual colonies can allow you to obtain a pure culture.




10

From CDC Lab Manual

M. tb on egg

medium

Liquid media

Liquid or broth medium has the advantage

of allowing detection of AFB more quickly

Drug susceptibility testing using growth in

liquid media leads to more rapid reporting

of results

Examples of liquid media are Trek and MGIT

systems

Inoculating growth media for culture (3)




11

MGIT Incubator




12

Accuracy problems in the TB lab

False positive results, due to:

o Cross-contamination during specimen

processing

o Specimen mix-up or mislabeling

Inadequate primary culture media (some

labs use only solid media)

Inaccurate drug susceptibility results due

to inadequate quality control




13

Identification of acid-fast bacilli (AFB)

Growth characteristics (preliminary ID)

Preliminary indication of M. tb can be made

from macroscopic & microscopic observation)

• Rate of growth

• Colonial morphology

• Pigmentation




14

Colonial

Morphology (1)

Smooth colonies

on 7H-10

medium




15

Rough colony

on 7H-10 medium

Colonial

Morphology (2)

Biochemical tests: not used now because too slow

There is a battery of 8-12 biochemical tests used

to differentiate within the mycobacterium genus

Nitrate reduction and niacin accumulation are

definitive for M. tb

Identification of acid-fast bacilli (AFB) (2)




16

Nucleic acid probe tests (non-amplified)

DNA probe tests are species or complex

specific

Require less time than biochemical tests for

identification

Commercial probes are available for M. tb

complex, MAC, M. kansasii and M. gordonae


High performance liquid chromatography (HPLC)

HPLC uses a chromatography method to identify

mycobacteria based on their mycolic acid profiles

(cell wall composition)

Instrument is expensive/usually reserved for

larger laboratories

MALDI-TOF (matrix assisted laser desorption

ionization-time of flight) is now more widely used than

HPLC

Like HPLC, expensive instrument, but quicker





17

Susceptibility testing of M. tuberculosis

When to test

All primary M. tb isolates from patients

should be tested

In addition, test isolates from relapse or

re-treatment cases

Test when drug resistance is suspected

Susceptibility testing of M. tuberculosis (2)Methods for susceptibility testing

• Agar proportion method compares growth on

agar media with and without one of the four

primary drugs

• Broth based (MGIT, Trek)

–Requires inoculation of the strain in broth

with each of the (5) primary drugs, plus

control vial

–Growth of the strain in a vial with a drug

indicates resistance to that drug




18




19

Direct detection of M.

tuberculosis in clinical material

Several commercial nucleic acid amplification tests (NAAT) for M. tb are now available, including

• Gen-Probe Amplified Mycobacterium Tuberculosis Direct (AMTD) (for smear + or smear -)

• Cepheid GeneXpert (now has FDA clearance)

These tests are designed to amplify and detect DNA specific to M. tb

The sensitivity of these methods allows for direct detection of M. tb in clinical specimens

Uses of NAAT for Direct Detection of MTBC in Respiratory Specimens

(NAAT = nucleic acid amplification test)

CDC guidelines recommend as standard of practice

Will allow quicker diagnosis in some smear neg patients

Only if patients are true TB suspects

Only for untreated patients

Not necessary to test smear positives when classic TB

symptoms and history are present

Do test smear negatives when clinical suspicion of TB

is moderate or high




20

More encouragement to use NAAT

Pascopella (2004) J. Clin. Microbiol 42:4209

For smear neg patients, health care

providers often/typically don’t start Rx until

culture is +

Results in a delay in initiation of Rx, typically

3 weeks

NAA would detect many of these patients,

earlier initiation of Rx

MMWR guidelines: Jan. 16, 2009 58(1):7-10

• Spoligotyping (spacer oligonucleotide typing)

• MIRU/VNTR (mycobacterial interspersed repetitive units/ variable number of tandem repeats)

• RFLP fingerprinting (restriction fragment length polymorphism)

• Whole genome sequencing (WGS)

Genotyping methods




21

Spoligotyping summary

Gives a result as a number, so to tell if 2 strains are

different, just see if they have different numbers

Not too powerful at discriminating different strains.

Sometimes strains that are not part of the same

outbreak will have the same spoligotype—e.g.,

Manila strain & Beijing strain

Is now performed at CDC, using DNA sequencer

MIRU summary

A PCR-based method, like spoligotyping

Like spoligotyping, the result is a number (24 digits)

Uses a DNA sequencer instrument to analyze the

PCR products

Like spoligotyping, MIRU sometimes doesn’t

discriminate between unrelated strains

Since April 2009, a new 24 locus MIRU protocol is

in use, making it more powerfully discriminatory




22

Using MIRU and spoligotyping together

Both are PCR-based strain typing methods, so you

can do them with just a small amount of DNA

If 2 strains of M. tuberculosis are different from one

another, it is unlikely that they will have the same

spoligotype and the same MIRU type

Possible exceptions include Manila strains and

Chinese “Beijing” strains

Universal genotyping approach (at Michigan lab)

• All isolatesMIRU-VNTR

• Whole genome sequencing with Spoligotype inferredFrom sequence

∽ 2 weeks

Additional3 weeks




23

Genotyping: Uses

1. Cross contamination studies

2. Outbreak investigation

3. TB Control needs, such as identifying

settings where transmission occurs

Note: when genotyping links patients in a cluster,

but no epi links are found, whole genome

sequencing may be helpful in identifying which

patients are truly linked in the same chain of

transmission

Contact info for (Michigan) genotyping laboratory

James T. Rudrik, Ph.D. [email protected]

Office: 517-335-9641

Fax: 517-335-9631

Angie Schooley, B.S. [email protected]

Office: 517-335-9637

Fax: 517-335-9631

927 Terminal Drive Lansing, MI 48906

Please note that cultures for genotyping are

sent through the county public health laboratory.

mailto:[email protected]

mailto:[email protected]




24

Molecular Detection of Drug Resistance:

Resistance Mutations

GeneXpert &

Pyrosequencing

Principles of Molecular Detection of Drug Resistance

GeneXpert uses molecular beacon probes in an automated, user friendly (expensive) system

With molecular beacons: if normal susceptible gene targets are present, molecular beacon will hybridize and “light up”

Absence of molecular beacon signal suggests drug resistance

For sequencing (e.g. pyrosequencing), the actual gene sequence is determined

Actual sequence is more informative




25

Limitations of molecular detection of drug resistance

The predictive value of a negative result for INH (no

mutations found) is in the low 90s (about 93%) to date.

Pyrosequencing or GeneXpert can be used to guide

treatment until conventional drug susceptibility results are

available (usually about a month later).

Detection of a mutation in rpoB gene by GeneXpert

doesn’t always mean rifampin resistance

Some rpoB mutations don’t cause rif resistance

When GeneXpert detects a mutation, follow-up DNA

sequencing should be done to confirm rifampin resistance.

Molecular testing should be followed by culture-based DST

Drugs of interest:

For XDR screening

INH, RIF, KAN, AMK, CAP, fQs

Targeted Genes for pyrosequencing

katG, inhA promoter, ahpC, fabG for INH

rpoB for RIF

rrs for KAN, AMK & CAP

gyrA for Quinolones

GeneXpert detects resistance to rifampin only

49G Lin PSQ 11-2-10

Detection of Drug Resistance Mutations




26

Comparison of MB & PSQ

GeneXpert

MB

PSQ

Report Mutation present

or notExact SQ

Interpretation of silent

mutations or mutations

not conferring R

Misinterpret

as R

Does not

misinterpret

Hands-on time 1.5 hr, simple 2.5 hr, more steps

Total test time 2.5 hr 5 hrG Lin PSQ 11-2-10 50

Results show sequences.

You would know what mutations are if present.

More information provided and less ambiguity.

Silent mutations do not lead to wrong interpretation.

Mutations not conferring resistance do not lead to wrong

interpretation.

Difficult targets may still be sequenced.

At present, GeneXpert detects resistance to rifampin only.

51

Advantages of PSQ over MB




27

Suggestion for requesting molecular detection of

drug resistance

Acid-fast smear-positive specimen

Some of the specimen sediment is available for sending

to reference lab (State Microbial Diseases Lab or CDC)

Drug resistance is suspected, or

A susceptible population has been exposed, or

The culture is mixed or non-viable, so regular drug

suscept. testing can’t be done

CDC also has Molecular Detection of Drug Resistance

(MDDR) program: tests for mutations associated with

resistance to additional drugs—ethambutol,

pyrazinamide

Thank you

Documents

LABORATORY METHODS: TUBERCULOSIS DIAGNOSIS...Curry International Tuberculosis Center May 8-11, 2018 Laboratory methods: tuberculosis diagnosis TB Case Management and Contact Investigation