tb laboratory update · tb laboratory update learning objectives upon completion of this session,...

TUBERCULOSIS CASE MANAGEMENT AND CONTACT INVESTIGATION INTENSIVE

MARCH 19-22, 2019

TB LABORATORY UPDATE LEARNING OBJECTIVES

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

1. Utilize rapid identification methods for drug susceptibility for tuberculosis to improve patient

outcomes

INDEX OF MATERIALS PAGES

1. TB laboratory update – slide outline Presented by: Max Salfinger, MD, FAAM, FIDSA

41

SUPPLEMENTAL MATERIAL

1. Forbes BA, Hall GS, Miller MB, Novak SM, Rowlinson M-C, Salfinger M, Somoskövi A,

Warshauer DM, Wilson ML. 2018. Practice guidelines for clinical microbiology laboratories:

mycobacteria. Clin Microbiol Rev 31:e00038-17. https://doi.org/10.1128/CMR.00038-17

2. Salfinger M. Molecular Assay Testing to Rule Out Tuberculosis—Be That Early Adopter. JAMA

Intern Med. 2018;178(10):1388–1389. doi:10.1001/jamainternmed.2018.3628

3. Baldwin SL, Larsen SE, Ordway D, Cassell G, Coler RN (2019) The complexities and challenges

of preventing and treating nontuberculous mycobacterial diseases. PLoS Negl Trop Dis 13(2):

e0007083. https://doi.org/10.1371/journal.pntd.0007083

4. Peloquin C. 2017. The role of therapeutic drug monitoring in mycobacterial infections.

Microbiol Spectrum 5(1):TNMI7-0029-2016. doi:10.1128/microbiolspec.TNMI7-0029-2016

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.

https://doi.org/10.1128/CMR.00038-17

https://jamanetwork.com/journals/jamainternalmedicine/fullarticle/2697836?utm_campaign=articlePDF&utm_medium=articlePDFlink&utm_source=articlePDF&utm_content=jamainternmed.2018.3628

https://doi.org/10.1371/journal.pntd.0007083

http://dx.doi.org/10.1128/microbiolspec.TNMI7-0029-2016

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


MARCH 19-22, 2019

• 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 detection 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.

• 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.


MARCH 19-22, 2019

• 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.

TB Laboratory UpdateMax Salfinger, MD, FIDSA, FAAMUniversity of South Florida, College of Public Health

TB Case Management and Contact Investigation IntensiveMarch 19-22, 2019 1

SCHOOL OF PHYSICAL THERAPY & REHABILITATION SCIENCES UNIVERSIT Y OF SOUTH FLORIDA

COLLEGE OF PHARMACYUNIVERSIT Y OF SOUTH FLORIDA

COLLEGE OF NURSINGUNIVERSIT Y OF SOUTH FLORIDA

COLLEGE OF PUBLIC HEALTHUNIVERSIT Y OF SOUTH FLORIDA

UNIVE RSIT Y OF SOUTH F LORIDA

MORSANI COLLEGE OF MEDICINE MORSANI

COLLEGE OF MEDICINE







COLLEGE OF MEDICINE

Our Practice Is Our Passion

TB Laboratory Update

Max Salfinger, MD, FIDSA,FAAMCo-Lead, DrPH Laboratory Concentration

University of South Florida, College of Public Health

[email protected]

http://health.usf.edu/publichealth/







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


The journey sets the destination

1978-1981 University Hospital, Basel-Switzerland

1981-1992 University of Zurich, Dept. Medical Microbiology

1986-1988 Sabbatical – Denver, Colorado

National Jewish, University Hospital, Webb-Waring Lung Institute

1992-2006 New York State Department of Health, Albany, New York

2006-2012 Florida Department of Health, Tallahassee, Florida

2012-2018 National Jewish Health, Denver, Colorado

2018 – present University of South Florida, College of Public Health

Tampa, Florida

mailto:[email protected]

http://health.usf.edu/publichealth/









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction

❑Direct Detection (NAAT)

❑Decision to Discontinue Airborne Infection Isolation in Healthcare Settings

❑Acid-fast Bacilli (AFB) Smear Microscopy

❑Growth Detection

❑Identification (incl. NTM)

❑Antimicrobial Susceptibility Testing (AST)

❑Genotyping

❑Systems / Algorithms







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Reported Tuberculosis (TB) Cases

United States, 1982–2016 [CDC]









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB Elimination

Goal: One TB case per 1 Million Pop.

Cases Cases

Actual Goal Factor

2017 US 9,093 323 28

Arizona 188 8 24

Colorado 84 6 14

New Mexico 37 3 12

Utah 29 4 7







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Mycobacterium spp.

Source:

http://www.bacterio.net/mycobacterium.html

Number of species cited in this file: 198

Number of subspecies cited in this file: 14

http://www.bacterio.net/mycobacterium.html









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Pulmonary NTM – Medicare Beneficiaries

AJRCCM 2012 Adjemian et al







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Burden of Pulmonary NTM Disease - US

Strollo et al. The Burden of Pulmonary Nontuberculous Mycobacterial Disease in the United States. Ann Am Thorac Soc Vol 12, No 10, pp 1458–1464, Oct 2015

Measurements and Main Results: In 2010, we estimated 86,244 national cases, totaling to $815 million, of which 87% were inpatient related ($709 million) and 13% were outpatient related ($106 million). Annual state estimates varied from 48 to 12,544 cases ($503,000–$111 million), with a median of 1,208 cases ($11.5 million). Oceanic coastline states and Gulf States comprised 70% of nontuberculous mycobacterial disease cases but 60% of the U.S. population. Medical encounters among individuals aged 65 years and older ($562 million) were twofold higher than those younger than 65 years of age ($253 million). Of all costs incurred, medications comprised 76% of nontuberculous mycobacterial disease expenditures. Projected 2014 estimates resulted in 181,037 national annual cases ($1.7 billion).









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Laboratory’s charge

To provide the

clinician with

accurate results in

a timely fashion







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Toolbox 1

✓ Specimen – sputum, CSF, formalin-fixed tissue – NALC-NaOH versus Oxalic acid (CF w/history of Pseudmonas

aeruginosa)

– AFB microscopy

– Solid (NTM plate) & broth-based media

– NAAT-D (TB complex, NTM - mostly MAC)

– NAAT-R (RIF, INH and more)

– Direct AST

✓ Patient management (culture negativity after 2 months on treatment)

Ideally, molecular TB testing 7 Days a week









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Toolbox 2

✓ AFB positive culture (broth-, solid-based media)

– TB Yes/No (final identification within TB complex)

– NAAT-R

– Broth-based AST

– Agar-based AST

– Minimal Inhibitory Concentration (MIC)

✓ Population management/genotyping

– RFLP-IS6110, Spoligo and MIRU

– whole genome analysis

– standardization through contracted PHL-MI







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Quality Specimen

Quality testing

requires

quality specimen

[5 to 10 ml sputum]









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Quality Specimen

Sputum, expectorated or induced:

Collection: Instruct patients on the proper method of sputum collection

– the material brought up from the lungs after a productive cough what is desired, and not nasopharyngeal discharge and saliva

• 5 - 10 mL sputum collected in a sterile container.

• Difficulty in producing sputum– sputum induction by inhalation of an aerosol of sterile hypertonic

saline (3%) or sterile water produced by a nebulizer that causes coughing. Label as INDUCED

• Perform in areas with adequate environmental controls under supervision.

• 3 consecutive specimens in 8- to 24-hour intervals, with at least one being an early morning specimen.

• Sputum specimens should not be pooled.







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Quality Specimen

CSF:

• Collection: At least 5 mL of CSF should be aseptically collected.

• Minimum volume required: 2 to 3 mL; optimal volume is 10 mL.

• A separate sample should be collected for chemistry and hematology.

Gastric Lavage:

• Collection: Specimens should be collected in early morning before patients eat and while they are still in bed. The lavage should be performed with 25 to 50 mL of chilled, sterile, distilled water. Recovered sample should be placed in a leak-proof, sterile container (e.g., 50-mL conical tube).

• Transport: Gastric wash or lavage material should be submitted in a sterile leak-proof container, such as a sterile 50-mL conical tube or sterile urine collection container.

• Transport time and temperature: Specimens should be transported at room temperature as soon as possible.

– If transport is delayed for more than one four hour, specimens should be neutralized with 100 mg sodium carbonate within one hour of collection, and transported as soon as possible at room temperature.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Quality Specimen

Abscess:

• Tissue (at least 1 g, if possible) or fluid is preferred. Tissue should not be frozen or preserved.

• A swab is strongly discouraged unless it is the only specimen available. Swabs should be submitted in 2 to 3 mL sterile saline. Swabs submitted in transport medium or a commercial swab transport device are unacceptable.

Blood:

• Collection: Manufacturer’s instructions for automated blood culture systems should be followed.

• Alternatively, 10 mL whole blood should be collected aseptically in a yellow-top collector tube containing SPS, or green-top collector tube containing heparin.

• Blood must not be collected in a red-top tube, EDTA (purple top), or ACD (yellow top).

• Minimum volume is 5 mL for adults; 1 mL for children.







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping










COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB NAAT

• FDA approved for respiratory specimens‾ Smear-positive (Dec. ‘95)‾ Smear-negative (Sept. ‘99)

• MMWR, January 16, 2009 [Universal]

In July 2013, the FDA granted Market Authorization to a cartridge-based assay. This NAA test can simultaneously identify Mycobacterium tuberculosiscomplex (TBC) and genetic mutations associated with resistance to rifampin from raw sputum and concentrated sputum sediments.







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB NAAT Recommendations

“NAA testing should be performed on at

least one respiratory specimen from each

patient with signs and symptoms of

pulmonary TB for whom a diagnosis of TB is

being considered but has not yet been

established, and for whom the test result

would alter case management or TB control

activities.” MMWR Jan 16, 2009









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Cartridge-based NAAT

Boehme CC et al. N Engl J Med 2010;363:1005-1015







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


US Study - California

217 clinical specimens/suspicion of TB

• 98% AFB smear pos.; culture pos.

• 72% AFB smear neg.; culture pos.

• 41 NTM negative

• 1 M. bovis positive

J Clin Microbiol 49:1621-23 (2011)









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Xpert only performed

… the following actions must be taken:

• It is strongly recommended that specimen be sent to a reference

laboratory for AFB smear and culture as soon as possible

regardless of the NAA result. If there is a sufficient volume of raw

sputum, split the specimen and send to a reference laboratory for both

concentrated AFB smear and culture. The sample must be split prior to

the laboratory mixing a sputum sample with the Sample Reagent (or

SR). If volume is insufficient, request an additional sputum specimen for

AFB smear and culture.

• Report results from a cartridge-based assay as soon as available while

awaiting culture confirmation.

• If RIF resistance is detected, a specimen should be sent to a

reference laboratory to confirm the resistance by DNA sequencing

as soon as possible.

APHL Factsheet Sept 2013







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB NAAT Comparison

AFB Smear + Smear -

MTD* 97 76

Laboratory Developed Test** 99.6 75.4

Xpert*** 100 71.7

Xpert – Ultra**** -&+/+ 90 63

Xpert**** -&+/+ 77 46* Greco et al Thorax 61:783-790(2006)

** Halse et al JCM 48:1182-1188(2010)

*** Helb et al JCM 48:229-237(2010)

**** Dorman et al Lancet ID18:76-84(2018)

Ultra: CE-marked – Not FDA approved









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB NAAT - CSF

• HIV-infected adults with suspected

meningitis at Mbarara Regional Hospital

(Uganda). Centrifuged CSF, resuspended

the pellet in 2 mL of CSF, and tested 0·5

mL with mycobacteria growth indicator

tube culture, 1 mL with Xpert, and

cryopreserved 0·5 mL, later tested with

Xpert Ultra.Bahr et al Lancet Infect Dis. 2018 Jan; 18(1): 68–75.







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB NAAT - CSF

• Xpert Ultra sensitivity was 70% (95% CI 47–87; 16 of 23 cases) for probable or definite tuberculous meningitis compared with 43% (23–66; 10/23) for Xpert and 43% (23–66; 10/23) for culture.

• Testing 6 mL or more of CSF was associated with more frequent detection of tuberculosis than with less than 6 mL (26% vs 7%; p=0·014).

Bahr et al Lancet Infect Dis. 2018 Jan; 18(1): 68–75.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Decision to Discontinue Airborne

Infection Isolation in Healthcare Settings

NTCA/APHL Consensus Statement on the Use of Cepheid Xpert MTB/RIF Assay in Making Decisions to Discontinue Airborne Infection Isolation in Healthcare Settings

• It is important to note that the process described herein is not to be used alone to rule out TB; Xpertnegative or acid-fast bacilli (AFB) smear-negative sputum may contain viable organisms and represent infectious tuberculosis.

• Furthermore, NAA testing should not be used to monitor response to treatment or to release a newly confirmed TB patient from A.I.I.

April 2016









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE



Infection Isolation in Healthcare Settings







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE



Infection Isolation in Healthcare SettingsInterpretation of an Xpert result must be made in the context of

the clinical and radiographic presentation and the clinician’s

suspicion for infectious TB. A decision to remove a patient

with a negative Xpert result from AII must consider the

clinical presentation and the risk of possible transmission

of TB from an infectious patient to others. Such a decision

should not be based on sputum test results alone. The

sensitivity of sputum testing for TB is subject to variability from a

variety of factors, including sampling (e.g., poor specimen

quality), inappropriate transport and processing of the specimen,

errors in performance of the assay itself, and errors in labelling

or reporting.

NTCA/APHL GeneXpert Consensus Statement – April 2016









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


San Francisco study

• In a prospective cohort study with a pragmatic, before-and-after implementation design, the authors analyzed 621 consecutive hospitalized patients undergoing sputum examination for evaluation of active pulmonary TB from January 2014 to January 2016 at the Zuckerberg San Francisco General Hospital and Trauma Center.

JAMA Intern Med. 2018; 178(10):1380-1388







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


San Francisco study

• The mean hospital costs per molecular TB test-negative patient decreased from $46,921 to $33,574 after implementation of the algorithm, providing an average savings of $13,347 per patient.

• The authors estimated utilization and costs for approximately 250 patients completing TB evaluation each year and projected a total annual savings to the hospital of $3.3 million.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE










COLLEGE OF MEDICINE







COLLEGE OF MEDICINE








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Reading/Interpretation: ZN & F Stain

A quantification of the numbers of acid-fast organisms per field should be rated 1+ to 4+. The number of tubercle bacilli in pulmonary secretions is directly related to the risk of transmission.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Demanding Instant Results!

20 Min 20 Hours









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Processing Sputum Samples

• Procedures kill all but 10-20% of the mycobacteria

• Contamination

2-5% of sputum specimens on Loewenstein-

Jensen medium (LJ)







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Growth Detection

• M. leprae – in armadillo for research; NOT in

clinical laboratories

• Suspicion for M. ulcerans, M. marinum, M.

haemophilum: additional media incubated at 30

to 32o Celsius

• Suspicion for fastidious organisms

(M. haemophilum, M. genavense, M.

paratuberculosis) require supplements – hemin,

mycobactin, etc.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Mycobacterium spp.

198 Species and 14 Subspecies in genus Mycobacterium as of March 18, 2019

M. tuberculosis complexM. tuberculosis; M. bovis; M. bovis BCG; M. africanum;

M. caprae; M. microti; M. canettii; M. pinnipedii; M. mungi; M. orygis









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


TB complex (2001-2004)

Wadsworth Center – NYS-DOH

Rapid and Simple Approach for Identification of Mycobacterium

tuberculosis Complex Isolates by PCR-Based Genomic Deletion

Analysis - Parsons et al JCM 40:2339 -2345 (2002)







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Human tuberculosis (N=35)

caused by

Mycobacterium bovis

New York City

2001 – 2004

Winters et al 2005 MMWR 54:605-608









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


M. bovis NYC 2001 - 2004







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Tuberculosis? 78 year-old Male

Somoskovi et al Eur J Clin Microbiol Infect Dis 26:937-940 (2007)









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Identification

• Nucleic acid probe kits• High Performance Liquid Chromatography

(HPLC) – cell wall• PCR Restriction Analysis (PRA)• Line Probe Assays• DNA sequencing• MALDI-TOF (Matrix-Assisted Laser Desorption

Ionization-Time of Flight) Mass Spectrometry –protein

• Whole Genome Sequencing (WGS)• Biochemicals are second







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Mycobacterial Species in Pulmonary NTM

Four integrated health care delivery systems*, 1991-2007

• M. avium complex 1,495 (80.1%)

• M. chelonae/abscessus 225 (12.1%)

• M. fortuitum 106 (5.6%)

• M. kansasii 102 (5.5%)

• M. simiae 53 (2.8%)

• M. xenopi 33 (1.7%)

*KP Southern California, KP Southern Colorado, Group Health, Geisinger

Am J Respir Crit Care Med 2010 Prevots et al.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


MALDI-TOF MS

MALDI-TOF MS can reliably and rapidly identify• Approximately 88% of Mycobacterium species, 90% of Nocardia species,

and 51% of other aerobic actinomycetes encountered in routine clinical practice at a tertiary medical center/reference laboratory. ✓ Using a custom, enhanced library and a streamlined extraction

procedure• Described the ability of the manufacturer’s library to identify these groups

of organisms and described the effects of lowering the accepted cutoff score from2.0 to 1.7✓ As the manufacturer continues to expand its database, many

laboratories will have the ability to identify many of the isolates they routinely encounter using MALDI-TOF MS.

✓ An expanded custom library may ultimately be the most useful tool for identification of the uncommon species encountered most often in a reference laboratory setting.

Buckwalter et al. J Clin Microbiol. 2016 Feb;54(2):376-84







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Notifiable NTM by State - 2016









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


NTM Reporting Requirements

State Reporting Time for NTM What is Required to be Reported (websites accessed on 12-11-2016)

Maryland within one working day Mycobacterium spp., other than Mycobacterium tuberculosis complex or

Mycobacterium leprae

Mississippi one week Nontuberculous mycobacterial disease

Missouri within 3 days Nontuberculosis mycobacteria (NTM)

Nebraska within 7 days Mycobacteria spp. (including M. tuberculosis complex organisms [for genotyping]

and all “atypical” species, to include culture, nucleic acid tests, or positive

histological evidence indicative of tuberculosis infection or disease)

Nevada not specified Submission of isolates of Mycobacterium spp.

New Jersey within 72 hours Mycobacterium, atypical

New Mexico within 24 hours Tuberculosis or other nontuberculous mycobacterial infections (including

Mycobacterium avium complex or leprosy)

Ohio close of the next biz. day Mycobacterial disease other than tuberculosis (MOTT)

Oregon one working day Nontuberculous mycobacterial infection (nonrespiratory)

Virginia immediate Results of cultures positive for any member of the Mycobacterium tuberculosis

complex (i.e., M. tuberculosis, M. bovis, M. africanum) or any other mycobacteria.

Results of rapid methodologies, including acid hybridization or nucleic acid

amplification, which are indicative of M. tuberculosis complex or any other

mycobacteria.

Wisconsin within 72 hours Mycobacterial disease (nontuberculous)







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


WGS - NYSComprehensive Whole-Genome Sequencing and Reporting of Drug Resistance

Profiles on Clinical Cases of Mycobacterium tuberculosis in New York State

ABSTRACT Whole-genome sequencing (WGS) is a newer alternative for

tuberculosis (TB) diagnostics and is capable of providing rapid drug resistance

profiles while performing species identification and capturing the data

necessary for genotyping. Our laboratory developed and validated a

comprehensive and sensitive WGS assay to characterize Mycobacterium

tuberculosis and other M. tuberculosis complex (MTBC) strains, composed of a

novel DNA extraction, optimized library preparation, pairedend WGS, and an in-

house-developed bioinformatics pipeline. This new assay was assessed using

608 MTBC isolates, with 146 isolates during the validation portion of this study

and 462 samples received prospectively. In February 2016, this assay was

implemented to test all clinical cases of MTBC in New York State, including

isolates and early positive Bactec mycobacterial growth indicator tube (MGIT)

960 cultures from primary specimens.

J Clin Microbiol. 2017 Jun; 55(6): 1871–1882.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


WGS – added value

✓ Faster turn-around time

✓ More comprehensive results

❖ Detect mixed infections

❖Many predictors of drug resistance

❖ Emerging resistance

✓ Cost effective

❖ Replace existing assays (real-time

PCR, pyrosequencing,

spoligotyping)

❖ Staff time savings







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


WGS to diagnose tuberculosisPosted on March 29, 2017 at 12:46 pm

• Public Health England has announced that Whole Genome Sequencing (WGS) is now being used to identify different strains of tuberculosis (TB).

• This is the first time that WGS has been used as a diagnostic solution for managing a disease on this scale anywhere in the world. The technique, developed in conjunction with the University of Oxford, allows faster and more accurate diagnoses, meaning patients can be treated with precisely the right medication more quickly. Where previously it could take up to a month to confirm a diagnosis of TB, confirm the treatment choices and to detect spread between cases, this can now be done in just over a week by Public Health England’s Birmingham laboratory. This slows the spread of the disease and boosts the fight against anti-microbial resistance.

• This world first service has been developed in partnership with Genomics England, National Institute for Health Research (NIHR) and Wellcome Trust. The implementation of this technology will contribute to achieving the aims of the 100,000 Genomes Project.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping


Primary MDR TB, United States, 1993–2017*

0

0.5

1

1.5

2

2.5

3

0

50

100

150

200

250

300

350

400

450

1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017

Year

Number of cases Percentage of total casesPercentageNo. of cases

* Based on initial isolates from persons with no prior history of TB; multidrug-resistant TB (MDR TB) is defined as resistance to at least isoniazid and rifampin.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Reference AST

Agar Proportion Method [CLSI M24]:▪ % resistant colonies

▪ Recognition of mixed cultures

▪ Up to 3 weeks’ incubation

▪ Direct AST (AFB+ smears)

• Broth-based methods [WHO]:▪ Susceptible vs. resistant

▪ Shorter TAT

▪ Walk-away system

▪ Strains with elevated MICs under-recognized







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Advantages of MIC

• Microtiter platform for first- and second-

line anti-TB drugs available

• Multiple concentrations tested – internal

control

• MIC will detect strains with altered

susceptibilities

• Value of MIC result is enhanced by the

result of the therapeutic drug monitoring









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


CLSI M24

M24 recognizes agar proportion as the reference methodology on which all other methodologies are based. In addition, this standard includes recommendations for using commercial broth susceptibility methods with shorter incubation times, which are now in widespread use for MTBC susceptibility testing, and information on molecular methods for detecting drug resistance and their integration with culture-based methods.







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


CLSI M24 MGIT & VersaTrek









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


CLSI M24 7H10, 7H11, LJ







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Molecular testing

Drug Gene Sens. Spec.

RIF rpoB 97.1 97.4

INH katG, inhA 86.0 99.1

EMB embB 78.8 94.3

PZA pncA 86.0 95.9

F-quinolones gyrA 79.0 99.6

Curry Center: Drug-resistant tuberculosis – A survival guide

for clinicians, 3rd ed. 2016









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Molecular Testing

Current methods for detection of drug resistance in

M. tuberculosis complex:

Cepheid Clin. & Conc. 2.5 h RIF

Hain Conc. & Cult. 6-7 h RIF/INH,oth.

Pyroseq. Conc. & Cult. 5-6 h RIF/INH,oth.

Sanger seq. Conc. & Cult. 1-2 days RIF/INH,oth.

Curry Center: Drug-resistant tuberculosis – A survival guide for

clinicians, 3rd ed. 2016







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Molecular Testing - Limitations

• Potential to identify mutations that do not confer phenotypic resistance

• Not all genetic loci associated with resistance are known; therefore,

‘no mutation detected’ does not

rule out resistance









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Frequency of Silent rpoB Mutations

Kuwait 1/12 8%

(JCM April 2015)

Haiti 2/153 1.3%

(PLOSOne Sept 2014)

Spain 2/8 25%

(JCM Oct 2011)

California PHL 26/154 17%(pers. comm.)

Texas PHL 3/19 16%(pers. comm.)







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Requesting molecular detection of drug

resistance• Acid-fast smear-positive specimen

• Some of the specimen sediment is available for sending to reference lab (State Public Health Lab)

• Drug resistance is suspected, or

• A susceptible population has been exposed, or

• The culture is mixed or non-viable, so regular antimicrobial susceptibility 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









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping








COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Genotyping Methods

• 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)









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Spoligotyping

• 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







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


MIRU

• 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









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


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







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Universal Genotyping Approach (MI Lab)

• All isolates

MIRU-VNTR

• WGS with

spoligotype inferred

from sequence









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Contribution of Genotyping

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

National Tuberculosis Genotyping Surveillance Coverage* by Year: United States†, 2004–2017

52.6

68.4 70.1

80.8 81.686.9

91.694.2 94.9 95.9 96.7 97.1 97.4 96.3

0

10

20

30

40

50

60

70

80

90

100

2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017

Pro

po

rtio

n o

f cu

ltu

re c

on

firm

ed T

B

case

s ge

no

typ

ed (

%)

* The proportion of positive cultures with at least one genotyped isolate.† Includes 50 states and the District of Columbia.§ For the year 2020, the national goal for TB genotyping surveillance coverage will change to 100%.

National Goal, 94%§



Number of County-based Tuberculosis Genotype Clusters* by Cluster Size, United States, 2015–2017

*Genotype cluster is defined as two or more cases with matching spoligotype and 24-locus MIRU-VNTR (GENType) within a county during the specified 3-year time period.

928

229

11046 22 17 11 10 36

0

200

400

600

800

1,000

2 casecluster

3 casecluster

4 casecluster

5 casecluster

6 casecluster

7 casecluster

8 casecluster

9 casecluster

≥10 case cluster

Nu

mb

er o

f cl

ust

ers

Number of Persons in Cluster







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Topics

❑Introduction




❑Growth Detection



❑Genotyping










COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Practice Guidelines for Clinical

Microbiology Laboratories: Mycobacteria







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


In-house AFB service performed -

APHL/CDC Survey 2011

100%

82%

37%









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Healthy People 2010, 2020, 2030?

• IID-32 Increase the proportion of culture-confirmed TB

patients with a positive nucleic acid amplification test

(NAAT) result reported within 2 days of specimen

collection

• Baseline: 32.0 percent of culture-confirmed TB patients with

a positive nucleic acid amplification test (NAAT) had their test

results reported within 2 days of specimen collection in 2008

• Target: 77.0 percent

• Target-Setting Method: Maintain consistency with national

programs, regulations, policies, and laws.

• Data Source: National TB Surveillance System (TB),

CDC/NCHHSTP







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Healthy People 2020 – status I

In 2015, the performance toward the

objective was 46% in public health

laboratories; no data were collected for non-

public health laboratories.









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Healthy People 2020 – status II

During the postimplementation period,

clinicians at the Zuckerberg San Francisco

General Hospital and Trauma Center

ordered a NAAT on 75% of possible TB

patients in airborne infection isolation.

However, California data are lower, with

30% receiving a NAAT in 2017 (but a

significant increase from 9% in 2010).







COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


March 24 – World TB Day









COLLEGE OF MEDICINE







COLLEGE OF MEDICINE


Maroon Bells, Colorado

Roseate Spoonbills, Florida

tb laboratory update · tb laboratory update learning objectives upon completion of this session,...

Documents