Microbiological diagnosis of TB

José Domínguez1 and Sabine Rüsch-Gerdes2

1Servei de Microbiologia. Fundació Institut en Ciències de la Salut Germans Trias i Pujol. Badalona. Spain

2 Forschungszentrum Borstel, National Referencelaboratory for Mycobacteria. Borstel.

Germany

Microbiological diagnosis of TB:Detection, identification and

molecular epidemiology

José DomínguezServei de Microbiologia.

Fundació Institut en Ciències de la Salut Germans Trias i Pujol

One of the main objectives for TB control

“Diagnosis, diagnosis & diagnosis”William Osler

HistologyClinical suspicion Microbiology

Objectives for TB control

•To rapidly diagnose patients with active TB and treat them correctly.

•To have rapid diagnostic methods, with high sensitivity and specificity to diagnose diseased patients at the beginning of the symptoms for an adequate treatment prescription

Diagnosis of tuberculosis

Latent Infection Active tuberculosis

Smear examination

Solid and liquid culture

Identification

Susceptibility testing methods

TST

IFN- techniques

MolecularEpidemiology

RFLP

Spoligotyping

MIRU

Molecular methods-Detection-Identification-Detection of resistance

Clinical samples

Samples!! Respiratory and extra-respiratory

Quantity: high inoculums means fast growthQuality, including sputum, high yieldLocalization, biopsies when possible

Rapid shipmentPrevious to starting treatment

Think of histology

The most important:Clinical-Microbiologist-Pathologist Communication

Decontamination

• Eliminate normal flora from the non-sterile samples (micobacteria is acid and alkaline resistant)• Homogenization to release the bacteria from the sample and allow access to the nutrient present in the media

i.e. KubicaN-acetyl-cysteine: homogenizationNaOH: decontaminantNeutralization by phosphate buffer

Homogenization

Sample mixing

Phosphate buffer

Centrifugation

Pellet

Smear microscopy

Ziehl-Neelsen stain Auramina O stain

Fast; Cheap; Monitorization of treatment; Low sensitivity

Hospital Univ. Germans Trias i Pujol 2004 -2007 Pulmonary Disseminated Extrapulmonary*

TOTAL 125 18 60

Positive smear 83 (66.4%) 11 (61.1%) 11 (18.3%)

Negative smear 42 (33.6%) 7 (38.9%) 49 (81.7%)

*Adenopathy, 4/28 (14.2%); Pleural, 1/17 (5.9%)

Decontamination (in non sterile samples)

Culture in the adequate media

Culture in solid and liquid media

• Sometimes the only place where the mycobacteria can be isolated• Gold standard• Molecular epidemiology• Drug susceptibility testing

DST

Growth in solid media

Slow: 15d-2mDivision time 18h

Growth in liquid media7-42d

Inoculums!!

Identification Classical and Molecular

methods

Identification molecular methods

InnoLiPA MycobacteriaAccuProbe

GenoType Mycobacterium CM/AS, GenoType MTBC

Identification molecular methods

Sequencing

PRA (Polymorphism Restriction Amplification)

Amplification, by PCR of a fragment of the hsp65 gen, followed by a restriction with 2 restriction enzymes (BstEII y HaeIII).

Pyrosequencing

PPi

ATP

M.tuberculosis detection in clinical samples by molecular methods

Method Target Detection methodSensitivity in respiratory samples (%)

Sensitivity inextra respiratory

samples (%)

Overall specificity

(%)

AMTD2 16S rRNA Chemiluminometric 80-100 60-90 95-100

LCx b antigenic protein Fluorimetric 80-90 65-80 90-100

AMPLICOR 16S rRNA Colorimetric 75-100 45-60 90-100

BD ProbeTecIS6110 and16S rRNA

Fluorimetric 55-100 30-80 45-100

INNO-LIPA v2 IR16S-23S Colorimetric 50-95 60-80 90-100

GenoType Direct

23S rRNA Colorimetric 60-95 60-80 95-100

PCR real time 16S rRNA Fluorimetric 70-90 65-85 85

* In smear negative samples the sensitivity is reduced in a 50%

Role of the clinical suspicion level inthe evaluation of the molecular methods

Catanzaro A. et al JAMA 2000

Problem: there are positive results in negative smear and culture samples.

• RFLP (IS6110)

• Spoligotyping

• MIRUs

Molecular epidemiology

• Insertion sequence present exclusively in the M.tuberculosis complex: IS6110

• High polymorphism between no related strains regarding the number of copies and their localization in the chromosome.

• Advantages: High discriminative power.• Disadvantages: Slow, laborious and with

certain complexity.

Restriction Fragment Length Polymorphism (RFLP)

Electrophoresis

Pvu II

Extraction and restriction

Hybridization

Transference

Radiographic develop

• The DR sequences (direct repeat) are repeated sequences of 36 bp in only one locus of the M.tuberculosis chromosome, separated by sequences of 34 to 41 bp.

• The technique is based on a PCR of the locus where the DR sequences are located. The amplification product is hybridized with oligos synthesized from the inter-DR spaces.

• The presence or absence of different DR allows a specific pattern for each strain.

• Advantages: Few DNA is required, easy interpretation• Disadvantages: Lesser discriminative power than the

RFLP.

Spacer oligonucleotype typing (Spoligotyping)

• Determine the number of repetitive units in 12 (15 or 20) different locus of one genetic sequence called “mycobacterial interspersed repetitive units (MIRUs)”. The number of repetitions is detected by PCR.

• The number of repetitive units in each locus is calculated by the size of the fragment amplified with the specifics primers.

• MIRU-VNTR is more discriminative than the spoligotyping and similar to the RFLP-IS6110.

• Advantages: rapid, simple and automatic.• Disadvantages: In study

Micobacterial Interspersed Repetitive Units (MIRU)

Micobacterial Interspersed Repetitive Units (MIRU)

MIRU 40 MIRU 40

500

pb

1

4 32

• The microbiological diagnosis of TB will be rapid and accurate if adequate samples are collected and adequate inoculums are used. Don’t forget histology.

• The future of TB diagnosis remains in the application of new molecular techniques but, at the moment a cautious interpretation of the results is required.

• The sensitivity of the molecular tests vary, and is affected by the amount of bacteria present in the samples, and also by the clinical suspicion level. Low sensitivity is present in samples with low bacterial load, especially in extra respiratory samples.

• At the moment, new molecular methods can not substitute the conventional ones. The gold-standard is the culture, and the other methods have to be considered and interpreted as complementary diagnostic methods.

• Communication between clinicians and microbiologists is imperative.

Conclusions

National Reference Laboratory for Mycobacteria

Forschungszentrum Borstel

Sabine Rüsch-Gerdes

Microbiological Diagnosis of TBDrug Susceptibility Testing

Borstel 2010

Drug Susceptibility Testing

MDR TB – New Infection

WHO: MDR-TB & XDR-TB, The 2008 Report; February 2008

Drug Susceptibility Testing

For all TB strains

isolated, DST has to be

performed

Anteil resistenter Erreger in Deutschland,

2006

0

2

4

6

8

10

12

14

Quelle: RKI, Bericht zur Epidemiologie der Tuberkulose in Deutschland für 2006; 2008

MicroscopyCulture

NATDifferentiation

DST

Level 3 laboratory

MicroscopyCulture

NAT

BiostoffVerordnung (BioStoffV) 1999

Level 2 laboratory

Proportion method on Löwenstein-Jensen medium

H, R, E, S, PTH, CM, OFL, CS, NSA (instead of P)

Results available:4-6 weeks

No critical concentrations for new substances

Methods for Drug Susceptibility Testing

BACTEC 460TB All drugs except cycloserine

Methods for Drug Susceptibility Testing

Results available:1-2 weeks

Radioactive materials, waste

Results available:

1-2 weeks

Methods for Drug Susceptibility Testing

MGIT 960 For all drugs, except CS

Advantages compared to solid media:• more rapid• high quality of media• fully automated system• testing of 1st, 2nd, and new drugs• safety: plastic tubes

Disadvantages:• expensive• higher contamination rate• dependency on a company• no DST for Cycloserine

Liquid media compared to solid media

Infection control strategies

Proposed measures:

• Improved ventilation system• Reduced hospitalsation• Mask use• Isolation of patients• HIV-testing and therapy• Rapid drug-susceptibility testing

Time for the detection has a potential to reduce the extend of spread of resistant strains

Principle of the Line Probe Assays

Chromogen (MBT/BCIP)

Alkaline Phosphatase

StreptavidinBiotin

Nitrocellulose strip

DNA-probe

Biotin-labelled single stranded amplified target

Colour reaction

Control of the conjugate -

Amplification control - Amplification control

MTBC - Control rpoB -

rpoB Wild type 1 - rpoB Wild type 2 - rpoB Wild type 3 -rpoB Wild type 4 -rpoB Wild type 5 -rpoB Mut D516V -rpoB Mut H526Y -rpoB Mut H526D -rpoB Mut S531L -

Control katG -katG wild type -

katG S315T1 (ACC) -katG S315T2 (ACA) -

1 2 3 4 5 6 7 8

MTBDR – DNA Hybridisation Strip

1 2

Genotype® MTBDRplus Hybridization-Strip

1 RMPr INHr

2 RMPs INHs

fromCulture media

Results RMP+INH Resistance

100 % concordance between sequencing and MTBDR data

103 MDR strains

91 strains (88.4) with mutations in codon 315 of

katG

102 strains (99%) mutations in rpoB cluster

I

1 strain (1%) a mutation outside rpoB

cluster I

3 strains (2,9 %) with a mutation in inhA

+2 strains (1,9 %)

with a mutation in ahpC+

7 strains (6,8 %) with no mutation in katG, inhA and

ahpC

1 strain (1%) not detected as MDR (rpoB outside cluster I, ahpC)

+

+

+

1 2

Genotype® MTBDRplus Hybridization-Strip

1 RMPr INHr

2 RMPs INHsfrom specimens

Hillemann D, Rüsch-Gerdes S, Richter E.

Application of the Genotype MTBDR assaydirectly on sputum specimens.

Int J Tuberc Lung Dis 2006. 10:1057-1059.

Evaluation of the MTBDRplus Assay on Specimens

72 smear positive sputum specimens:

30 susceptible strains 32 MDR (RMPr/INHr) strains 10 INHr strains

Sensitivity RMP detection: 96.8%

INH detection: 90.2%Specificity RMP: 95.2%

INH: 100%

Line Probe Assays for DST

INNO-LiPA Rif TB

GenoType MTBDRplus

GenoType MTBDRsl

Resistance Sensitivity Specificity

RMP 98.1% 98.7%

INH 84.3% 99.5%

Ling et al., Eur Respir J 2008

Evaluated line probe assays

Molecular Basis

Drug Gene

Locus

Gene function Percent of

Resistance

Fluorochinolones gyrA DNA-Gyrase A appr.80-90%

Amikacin, Capreomycin, Kanamycin

rrs 16S rRNA appr. 80 %

tlyA Methylase

Ethambutol embB appr. 30-60%

• Overall sensitivity for OFL, AM, CM and EMB was 90.2 %, 83.3 %, 86.8 % and 59.0 %, respectively.

• Specificity was 100 % for FLQ, AM, and EMB, and 99.1 % for CM.

• Most prevalent mutations were:gyrA D94G in Oflr strainsrrs A1401G AMr/CMr strains embB M306V in EMBr strains

• The rapid detection of XDR strains is possible with the combined application of Lipas from DNA isolates and directly from sputum specimens.

Summary

Time-to-result: 1 h 45 min

GeneXpert

FIND 2009

Xpert MTB

Evaluation Partner Sites

FIND 2009

High tech for low tech settings:

Sensitivity and Specificity seems to be very good for the detection of TB and Rifampicin resistance.

Xpert MTB

Drug Susceptibility Testing

7 - 10 days

3 - 4 weeksSolid MediaLöwenstein-Jensen(Middlebrook)

Liquid MediaBACTEC 460 TBMGIT

Molecular based MethodsInnoLipaGenoTypeMTBDRXpert MTB‚home made‘- methods

Hours – 1day

Quality Control

internal external

Internal Quality Control

all reagents

all techniques

all staffs

All QC results have to be documented

Internal Quality Control

1 2 3 4 5 6 7 8 9 10 11

embB MUT1BembB MUT1A

embB WT1embB

rrs MUT2rrs MUT1rrs WT2rrs WT1

rrs

gyrA MUT3DgyrA MUT3CgyrA MUT3BgyrA MUT3AgyrA MUT2gyrA MUT1gyrA WT3gyrA WT2gyrA WT1

gyrA

TUBACCC

Drug Susceptibility testing

External Quality Control

Participation in international QA programs

Reliable results

To detect and treat patients properly