Exciting Developments Towards Non-Sputum Based Diagnosis of TB · Sandlund et al DMID 2018 HIV-...

Exciting Developments Towards Non-Sputum Based Diagnosis of TB

Niaz Banaei MDProfessor of Pathology and Medicine

Stanford University [email protected]

Active TB

Latent Infection

10 million cases estimated

1/4 of world’s population

Global Burden of M. tuberculosis

WHO 2018

Active TB

Latent Infection

10 million cases estimated6.4 million reported3.6 million diagnostic gap



WHO 2018

Contributors to Global Diagnostic Gap

Pediatric ExtrapulmonaryHIV/AIDSUnproductive

1 million 0.9 million

Active TB

Latent Infection

Incipient TB

10 million cases estimated6.4 million reported3.6 million global gap



WHO 2018

Recent and Projected Trends in Global TB Incidence Cases

Dye et al Annu Rev Public Health 2013

Active TB

Latent Infection and

Incipient TB

Sputum: Culture & NAATBlood: IGRA, RNA signature, biomarkersUrine: biomarkers (LAM)Breath: biomarkers

Blood: IGRA, RNA signature

Diagnostic Tools for M. tuberculosis

Active TB


Incipient TB

Blood: IGRA

Blood: IGRA


‘A 21st Century Solution for Latent TB Detection’

IGRAs entered the scene with a lot of promise

More sensitive and specific than TSTMore reproducible/objective

More predictive

Active TB

Sensitivity and Predictive Value of IGRAs

Pai et al. Clin Micro Rev 2014

Diel et al. Chest 2010

Sensitivity: 80-90s%Specificity: low due to LTBI

Latent Infection




Sensitivity: 40-70%Specificity: >90%Predictive value: <10%

Incipient TB




Sensitivity: 40-70%Specificity: low due to LTBIPredictive value: <10%

Active TB

Latent Infection

Incipient TB




Sensitivity: 80-90s%Specificity: low due to LTBI

Sensitivity: 40-70%Specificity: >90%Predictive value: <10%

Sensitivity: 40-70%Specificity: low due to LTBIPredictive value: <10%

‘A 21st Century Solution for Latent TB Detection’

IGRAs entered the scene with a lot of promise

More sensitive and specific than TSTMore reproducible/objective

More predictive

TB Ag Tube 1 (TB1): ESAT-6 and CFP-10 peptides for CD4 T Cells

TB Ag Tube 2 (TB2): ESAT-6 and CFP-10 peptides for CD4 and CD8

T Cells

QuantiFERON®-TB Gold Plus

TB1 +

TB2 +

+- +

-

-

-

• Interpretation of QFT-Plus using

manufacturer’s interpretation

Interpretation of QFT-Plus Results

• Evidence for role of CD8+ T cells in TB immunity

• IFN- positive Mtb-specific CD8+ T cells

– Associated with recent exposure to TB

– More frequently detected in active TB vs. latent infection

– Mycobacterial burden-dependent

– Detectable in active TB subjects with HIV co-infection

and young children

– Decline after anti-tuberculosis treatment

Why Target CD8 T Cells in QFT-Plus?

Does QFT-Plus have a higher sensitivity

than QFT-GIT for recent exposure to Mtb?

Barcellini et al ERJ 2016

Study Design

QFT-Plus vs. QFT-GIT

Prospective contact screening

Location: Milan, Italy

Contacts: Tested 119 adults with newly positive TST (≥5mm)

Included immunocompromised (9%)

Retested 10-12 weeks if negative

• QFT-Plus: 57.1% (68/119) vs. QFT-GIT: 47.1% (56/119)

QuantiFERON-TB Gold Plus performance in a high-risk population in the United States: a comparison with QuantiFERON-TB Gold In-tube, tuberculin skin test and T-SPOT.TBCDC Tuberculosis Epidemiologic Studies Consortium II (TBESCII)

Study Design

QFT-Plus vs. QFT-GIT

Prospective cross-sectional study

Location: 12 clinics in the U.S.

Contacts: Tested 508 individuals (adult and children) at high

risk for LTBI

Venkatappa et al JCM 2019

94%

99%

98%

Agree-

ment

QuantiFERON-TB Gold Plus performance in a high-risk population in the United States: a comparison with QuantiFERON-TB Gold In-tube, tuberculin skin test and T-SPOT.TBCDC Tuberculosis Epidemiologic Studies Consortium II (TBESCII)

Venkatappa et al JCM 2019

In 44 children with household

TB exposure, agreement

between the QFT-GIT and

QFT-Plus was 96%.


than QFT-GIT for recent exposure to Mtb?

Answer: No. Differences observed are

due to antigen formulation and not

immunological response.


than QFT-GIT in patients with active TB?


than QFT-GIT in patients with active TB?

QFT-GIT QFT-Plus P value TB Ag TB1 TB2 QFT-GIT QFT-Plus

Petruccioli et al

Tuberculosis 2017Italy Adult ≤7 days 0% 69 88% 90% <0.05 2.6 1.9 2.5 19 100% 100%

24 96% 96% >0.05

33 clin 85% 85% >0.05

Yi et al Sci Rep 2016 Japan Adult ≤14 days 4% 162 96% 96% >0.05 4.23 2.36* 2.85* 212 99% 97%

Horne et al IJTLD 2018 US & Japan Adult ≤14 days 2% 164 94% 93% >0.05 4.45 3.07* 3.56** ND --- ---

5 100% 100% >0.05 ND --- ---

7 clin 25% 25% >0.05 ND --- ---

87%4.67

Controls

77Germany 90%3.1* 3.7*

Sensitivity Specificity Country Age Abx Tx HIV+/ICH CasesMedian or Mean (IU/ml)

Hoffmann et al

Clin Microb Inf 2016Adult

Not

provided5%

Study

Kay et al AJTMH 2019 Eswatini Pediatric 0 days 42%Not

provided

Not

provided

Not

provided


than QFT-GIT in HIV+ patients with active

TB?

Study Design

Single arm

Prospective

Location: Zambia

TB Patients: Smear+ or Xpert+

68 HIV+

<3 days of anti-TB therapy

HIV-

HIV+

Prior study on QFT-GIT

Sensitivity: 63%

Active TB

Incipient TB

Blood: RNA signature

Blood: RNA signature

WHO 2018


Nature 2010

IFN I/II signalling and complement transcripts up 18 months

before TB diagnosis, while changes in myeloid , lymphoid,

monocyte and neutrophil responses occurred more proximally

Scriba PLoS Pathogens 2017

GBP5 promotes inflammasome assembly

DUSP3 regulator of JNK and ERK signalling

KLF2 anti-inflammatoryLANCET RM 2016

Genes comprising the 8

best signatures for

incipient TB

LANCET RM 2020

Diagnostic Accuracy of the 8 Best

Signatures for Incipient TB

LANCET RM 2020

LANCET RM 2020

Active TB

Incipient TB

Urine: biomarker (LAM)

WHO 2018


Urinary LAM Detection with Lateral Flow

(LAM)

Lipoarabinomannan (LAM)

Urinary LAM Detection with Lateral Flow

Alere Determine TB LAM Ag (AlereLAM)

≤100 101-200 >200CD4

Schiller Cochrane 2019

Sensitivity Specificity

≤100 101-200 >200CD4

WHO 2020

Novel Fujifilm SILVAMP TB LAM (FujiLAM)

Lancet Infect Dis 2019

Lancet Infect Dis 2019

Inpatients with HIV

OFID 2020

Inpatients and outpatients with HIV

Sensitivity 95%

Specificity 80%

2017

Active TB

Urine: biomarkersBreath: biomarkers

WHO 2018


Infectious Diseases Have Metabolic Signatures Need for Technological Innovation to Detect Them

Colorimetric Sensor Array

Urine

Breath

Colorimetric Array for Detection

of Volatile Signatures

• Digitally image before & after exposure & subtract.

After Exposure

ammonia

• Difference Map is a “molecular fingerprint”:

a unique 108-dimensional vector (36 ΔR, ΔG, ΔB).

(center avg.

300 pixels)

Difference Map

|Rafter- Rbefore|,|Gafter- Gbefore|,|Bafter- Bbefore|

• Printed array of chemically responsive dyes.

Before Exposure

Biggest color changesare boxed in gray.

9

mm

Urine collected

Gold StandardCases: Culture+/NAAT+Controls: Culture-/NAAT-

Sputum

Analysis

Difference

Diagnosis of Tuberculosis from Analysis of

Urine Volatile Organic Compounds

Highland Study

Correlation matrix indicates similarities between patients of the same category

TB

N=39

Non-TB

N=25

Sensitivity: 86.9%

Specificity: 87.6%

Lim et al ACS Sensors 2016

Urine collected

Kenya Study at KEMRI

TB: 200 Non-TB: 200

2 hr

Gold StandardCasesGeneXpert+ &or Cx+Controls: GeneXpert- & Cx-

Sputum

Analysis

Difference

Principal Component Analysis Score plots

Sensitivity: 78.3%

Specificity: 69.2%

Sensitivity: 57.9%

Specificity: 63.1%

Sandlund et al DMID 2018

HIV- Group HIV+ Group

Active TB

Blood: cfDNA Urine: cfDNA

WHO 2018


Application of cf DNA in Diagnostics

Fetal aneuploidy Cancer mutations Organ rejection

Microbial cf DNA

Infectious diseases

- EBVnasopharyngeal CA (Cancer Res 1999)

- Invasive fungal infection (CID 2013)

Biology of Cell-Free DNA

Discovered in 1948

(Mandel & Metais)

Removed by liver

Half-life 10-15min

Apoptosis: 180-1000bp

Necrosis: 10,000bp

Trans-renal

150-200bp

Target Population for cfDNA TB Diagnosis

Pediatric ExtrapulmonaryHIV/AIDSUnproductive

1 million 0.9 million

Accuracy of Plasma cf DNA for TB Diagnosis

IS611065%

(21/33)

93%

(18/19)

gyrB29%

(10/33)

100%

(19/19)Click et al

Scientific

Reports 2018

>18 yo Kenya PTB 43 ND PCR IS611044%

(19/43)ND

Brazil PTB 4974%

(34/49)

USA EPTB 875%

(6/8)

Stanford/GG/

IV>18 yo Uganda PTB 54 40 PCR IS6110

46%

(25/54)

100%

(40/40)

GWiS

Target Specificity

Digital

PCR

Cases Controls

Hogan et al

In

preparationPCR

PCR69

Method Sensitivity

Ushio et al

Tuberculosis

2016

Age

19

CountryTB

Type

53%

(8/15)

100%

(69/69)

33PTB

GWiS100%

(15/15)PTB 15 15

Vietenam

& SA>18 yo

>18 yo

Japan>18 yo

Bilateral vsUnilateral PTB

PTB+EPTB vs. PTB

↑cfDNA ↑cfDNA

Day14 Day0+1484% 94%

Accuracy of Urine cf DNA for TB Diagnosis

Miliary Multifocal LAN Pleural Joint90%(9/10)

67%(16/24)

72%(18/25)

33%(1/3)

45%(5/11)



vsRadiology vsSmear+ vsTTCxP Wk1 Wk12

écfDNA None None écfDNA 9/11Neg*100% with

retesting

Cannas et al

IJTLD 2008>18 yo Italy PTB 43 23

Nested

PCRIS6110

79%

(34/43)

100%

(23/23)

EPTB 82 70%

(57/82)

PTB 2518%

(5/25)

Labugger et al

Infection 2017>18 yo Germany PTB 11 8 PCR IS6110

64%*

(7/11)

100%

(8/8)

Stanford/GG/

IV>18 yo Uganda PTB 75 59 PCR IS6110

32%

(24/75)

98%

(58/59)

Fortun et al

IJTLD 2014

Sensitivity SpecificityTB

TypeTargetCases Method

TMA16S

rRNA

Not

Done>18 yo

CountryAge

0Spain

Controls


Pediatric ExtrapulmonaryHIV/AIDS

Potential of cf DNA in Diagnosis of TB

Cell-free DNA

in Urine

Accuracy

Sensitivity 40%-70%

Specificity ≈100%

Unproductive


Hogan

Preanalytical Variables Impacting

Pathogen cfDNA in Blood and Urine

Murugesan JCM 2019 PMID:31511335

Optimization of Variables Impacting Plasma cfDNA Detection


Comparison of Blood Collection Tubes


Comparison of Urine Collection Preservatives


Comparison of Blood Collection Tubes andUrine Preservatives in Patients with TB


Comparison of Plasma Processing Delay


Comparison of Urine Processing Delay


Comparison of 1 spin vs 2 spin Plasma Collection


Comparison of Whole vs 1 Spin Urine Processing


Comparison of Fresh vs. Frozen Plasma


Comparison of Fresh vs. Frozen Urine


Comparison of Plasma and Urine Volume

Preanalytical Variables Impacting

Pathogen cfDNA in Blood and Urine

Higher volume

more sensitive

EDTA is adequate


Up to 24 hr delay

is adequate

1 spin is adequate for plasma

No spin needed for urine

Freeze thaw

has no impact

Promega

OmegaQiagen

Thermo

Plasma

ccfDNA extraction

DNA spiking

Sample collection

tube

Processing delay

Centrifugation

Sample storage

Promega &Maxwell

KingFisher &Omega

KingFisher &Thermo

QiaSymphony &Qiagen


Mtb cfDNA Testing is Promising

Optimize extraction of cfDNA from plasma and urine

Short cfDNA fragments

At lower limit of detection

Assess sensitivity of TB cf-DNA using optimized pre-

analytics in adult and pediatric cohorts

Develop sample-to-answer assay

GeneXpert Ultra

Active TB


Incipient TB

Sputum: Culture & NAATBlood: IGRA, RNA signature, biomarkersUrine: biomarkers (LAM)Breath: biomarkers

Blood: IGRA, RNA signature


Acknowledgements

Stanford University

Kanagavel Murugesan

Rajiv Gaur

Fiona Senchyna

Hee-Won Moon

Catherine Hogan

Jason Andrews

Juan Santiago

Nobuyuki Futai

Global Good/IVL

UCSF/Uganda

Adithya Cattamanchi

Uganda Team

Stellenbosch University

Grant Theron

Funding

Stanford Global Health

Global Good/IVL, ChEM-H

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