Tuberculosis diagnostics

ปลอด TB ชีวีมีสุข แนวทางเพื่อการวินิจฉัยที่แม่นยำ รักษาได้ตรงจุด และได้ผลการรักษาที่ดี

S a n t i S i l a i r a t a n a , M D

D i v i s i o n o f Pu l m o n a r y M e d i c i n e , D e p a r t m e n t o f M e d i c i n e , Fa c u l t y o f M e d i c i n e Va j i r a H o s p i t a l

N a v a m i n d r a d h i r a j U n i v e r s i t y

Overview of Tuberculosis

Tuberculosis

20 * Diagnostics for tuberculosis: global demand and market potential *

1CHAPTER

WHO’s Global TB Monitoring and

Surveillance Project experts esti-

mate the total number of cases to

be 8.8 million (3.9 million sputum

smear-positives) (2).

In 2003, 4.1 million cases (1.9 mil-

lion sputum smear-positive) were

notified to public health officials

around the globe. Seven million of

the estimated 8.8 million cases are

concentrated in 22 high-burden

countries of the developing world

(Figure 2). If recent trends should

continue for the rest of this decade,

the projected global number of

new cases will increase to 10 mil-

lion cases in 2015 (3).

Exposure to TB

Subclinical "latent" infection

"active" TB disease

Sputumsmear

positive

Sputumsmear

negative

Drugsensitive

Drugresistant

Pulmonary

Smearpositive

Smearnegative

Drugsensitive

Drugresistant

Extrapulmonary

Fig

ur

e 1 TUBERCULOSIS CLASSIFICATION SCHEME

Fig

ur

e 2 THE ESTIMATED GLOBAL BURDEN OF TUBERCULOSIS

CUMMULATIVEINCIDENCE (%)

20%

35%

43%

47%

51%

54%

57%60%62%64%67%68%

70%72%73%

74%75%76%77%78%79%

80%

100%

ESTIMATEDINCIDENCE

1,788,0431,334,066

627,047362,819360,767278,392251,685241,537236,885195,207194,627160,688

144,942

137,260

110,319

106,20189,351

86,130

85,015

84,546

79,656

71,830

8,810,040

COUNTRY

IndiaChinaIndonesiaNigeriaBangladeshPakistanEthiopiaSouth AfricaPhilippinesKenyaDR CongoRussianFederationViet NamUR TanzaniaBrazilUgandaThailandMozambiqueZimbabweMyanmarAfghanistanCambodia GlobalEstimated TB Cases

Sou

rce:

refe

ren

ce 2

.More people die from TB than from any other curable infectious disease.

Every day 25,000 people develop active TB and 5,000 die of the disease.

WHO. Global tuberculosis control: surveillance, planning, financing: WHO report 2005. Geneva: WHO, 2005.

Poverty

Congregation

HIV pandemic

Tuberculosis-HIV Coinfection

* Diagnostics for tuberculosis: global demand and market potential * 53

resistant, it is estimated that over

400,000 people fall ill with MDR-TB

each year, and that over 50 million

people are latently infected with

MDR strains of TB (7). The regional

distribution of MDR-TB is illustrat-

ed in Figure 4. Two thirds of MDR-

TB cases occur in just three

countries, China, India and the

Russian Federation.

Another serious threat underlying

the need for improved diagnostics

is the HIV pandemic, which greatly

increases susceptibility to TB infec-

tion and disease, and decreases the

effectiveness of conventional diag-

nostic approaches. Globally, 12% of

new adult cases of TB are HIV co-

infected, but the burden of dual dis-

ease is concentrated in Africa and

in some regions in Asia (see Figure

5), where the collision between HIV

and high prevalence of latent TB

infection (50-90%) has sparked a

dramatic rise (3-10 fold in some

countries) in active TB cases.

In summary, tuberculosis is a glob-

al epidemic concentrated in the

developing world, in close associa-

tion with poverty and, increasingly,

HIV. Testing for tuberculosis

remains common in industrialized

countries, where immigrants make

up a large and growing fraction of

all cases. MDR-TB and HIV are both

substantial threats to TB control,

and have prompted significant

increases in expenditure on TB

diagnosis and treatment in devel-

oped countries since the mid-

1980s, when TB was declared to be

in the elimination phase in the

United States.

Global availabilityof TB laboratoryservices

Little information has been accessi-

ble on the availability of TB diag-

nostic services in developing

countries or the volume of testing.

To this end, we carried out a global

survey of TB laboratory services.

Surveys were distributed to 207

WHO Member States to gather

information on the number of pub-

lic and private laboratory facilities

performing sputum smear

microscopy, mycobacterial culture

and drug susceptibility testing

(DST). Information was also col-

lected on the volume of testing in

the public sector. Each survey of

the 116 survey responses was

screened and respondents were

contacted directly to explain errors

and/or unexpected information.

Raw data on the number of testing

centres for TB microscopy, culture

and DST are shown in Table 1.

Fig

ur

e 5 PREVALENT ADULT TB CASES COINFECTED WITH HIV, 2004

Sou

rce:

refe

ren

ce 3

.

Dye C, Watt CJ, Bleed DM et al. Journal of American Medical Association 2005; 293:2767-75.

The Gap between Estimated and Notified Cases

Estimated TB cases 8.8 Million

Health facility

TB casesDiagnostic

tests

Recorded & reported

4.1 Million cases reported

Detected but not notified private sector

military prisons

⊕⊖

WHO. Global tuberculosis control: surveillance, planning, financing: WHO report 2005. Geneva: WHO, 2005.

Multidrug-resistant and Extensively drug-resistant TB

Multidrug-resistant (MDR) TB Resistance against at least

rifampicin and isoniazid

Extensively drug-resistant (XDR) TB MDR-TB PLUS

Resistance to any fluoroquinolones AND

≥1 injectable second-line agents

O’Grady J, Maeurer M, Mwaba P et al. Current Opinion in Pulmonary Medicine 2011, 17; 134-141.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

(ethionamide, prothionamide, cycloserine, terizidone,para-aminosalicylic acid, clofazimine, amoxicillin-clavula-nate, clarithromycin, linezolid) is not recommended [12].Automated liquid culture systems are currently recom-mended by the WHO as the ‘gold standard’ for second-lineDST [12,13!!,14]. In this review, we describe the pheno-typic and genotypic methods currently available for thediagnosis of drug-resistant forms of M. tuberculosis anddiscuss future prospects for TB diagnostics.

Definitions of drug-resistant tuberculosisMDR-TB is defined as resistance to the two key first-lineanti-TB drugs, INH and RIF. XDR-TB is defined as TBcaused by strains of M. tuberculosis resistant to at leastINH and RIF (i.e. MDR-TB), plus any fluoroquinoloneand at least one of three injectable drugs used in anti-TBtreatment, capreomycin, kanamycin or amikacin [7!!,8!!].

Phenotypic drug susceptibility testingCulture-based or phenotypic DST methods are accurateand inexpensive but are disadvantaged by relying on thegrowth on M. tuberculosis, rendering them time consuming[15]. Phenotypic DST methods are performed on solid orliquid media as direct or indirect tests. Direct methods

are those used directly on patient samples where a setof drug-containing and drug-free media is inoculateddirectly with a patient specimen. Indirect DST involvesinoculation of drug-containing media with a pure culturegrown from the original patient specimen [12]. Commer-cial automated liquid culture DST methods have arelatively short turnaround time (because of sensitiveautomation and M. tuberculosis’s relatively faster growthin liquid compared with solid media) and are highlyaccurate but are expensive and require specialist equip-ment [16!]. The WHO considered evidence for theaccuracy and role of a number of noncommercial cul-ture-based methods that utilize widely available andinexpensive laboratory equipment and supplies andrecommended selected methods as interim measureswhile capacity for automated culture DST and/or geno-typic DST are being developed [1!!,17!!,18]. Microscopicobservation drug susceptibility (MODS) [17!!,19–21] andcolorimetric redox indicator (CRI) [22,23] methods and thenitrate reductase assay (NRA) [15,24!!,25] received WHOapproval [17!!]. Such methods have similar accuracy tocommercial liquid culture systems and could be imple-mented in high-burden, low-income settings with mini-mum cost; however, these tests require extensive operatortraining, standardization and quality assurance beforeimplementation [1!!].

136 Infectious diseases

Figure 2 Estimated percentage of multiple drug resistant tuberculosis among new tuberculosis cases, 2008a

, 0 to <3; , 3 to <6; , 6 to <12; , 12 to <18; , "18; ‘, no data available; , subnational data only. Reproduced withpermission from [2].

Diagnosis of Tuberculosis

AFB stain Myc Culture

Drug susceptibility

Chest radiography CT scan

History Chronic productive cough*

Sputum production* Prolonged low grade fever

Night sweats Weight loss

Physical examination Bronchial breath sound

Crepitation Digital clubbing

Establishing Tuberculosis: Pulmonary TB

Imaging

Additional test(s)

Clinical features suggestive for tuberculosis

Microbiology

Diagnostic Algorithm: Clinically-suggestive

Patient with clinical features suggestive for pulmonary tuberculosis

Sputum examination for acid-fast bacilli Chest radiograph

AFB - positive CXR - compatible with TB

AFB - negative CXR - compatible with TB

AFB - negative CXR - incompatible with TB

Sputum culture and drug susceptibility testing for mycobacteria Treatment for pulmonary tuberculosis

Look for alternative diagnosis

แนวทางเวชปฏิบัติการรักษาวัณโรคในผู้ใหญ่ พ.ศ. 2556 (ฉบับร่าง). สำนักวัณโรค กรมควบคุมโรค สมาคมอุรเวชช์แห่งประเทศไทย

Diagnostic Algorithm: Radiographically-suggestive

Asymptomatic patient withradiographically suggestive tuberculosis

Sputum examination for acid-fast bacilli Review previous chest radiograph

AFB - positive CXR - compatible with TB

AFB - negative CXR - unavailable

AFB - negative CXR - unchanged

Sputum culture and drug susceptibility testing for mycobacteria Treatment for pulmonary tuberculosis

Re-evaluation and repeat CXR in 3 months

AFB - negative CXR - active TB

AFB - negative CXR - old lesion

แนวทางเวชปฏิบัติการรักษาวัณโรคในผู้ใหญ่ พ.ศ. 2556 (ฉบับร่าง). สำนักวัณโรค กรมควบคุมโรค สมาคมอุรเวชช์แห่งประเทศไทย

Methods to Detect TB infection

Detecting TB Infection

Microscopic examination

Gene/molecular- based techniques

Mycobacterial culture

Immune reactivity detection

M. tuberculosis

detection

Sputum Microscopy for Acid-fast Bacilli

Friedrich Carl Adolf Neelsen (1854-1898)

Franz Ziehl (1857-1926)

Neelsen-Ziehl (Acid fast bacilli) Staining

Acid-fast bacilli appear pink in a contrasting methylene blue background

Diagnostic Threshold underly Light Microscopy

* Diagnostics for tuberculosis: global demand and market potential * 119

Socioeconomicconsequences of current TBdiagnostics

Figure 6 is a schematic diagram

of the timescale of a patient’s

encounters with both the TB bacillus

and the health care system. For a

variety of reasons, the patient’s

journey from the first appearance

of symptoms to the point at which

the patient is offered treatment

rarely follows a straight line.

The main reasons are:

1. The patient begins at a lower

level in the health care system

where TB diagnostic tests may

not be available.

2. Physicians and other health

care workers do not suspect TB

and do not order the appropri-

ate tests.

3. The operational demands of

the test are too great, leading

to non-compliance with the

test protocols.

4. The technical performance

of the test is suboptimal.

One or more of these factors

contribute to missed or delayed

TB diagnosis, which can have

important economic and medical

consequences. A critical economic

burden imposed by misdiagnosis

is that substantial resources of the

patients and the health care system

can be used up before a definitive

diagnosis is obtained. There are

three main consequences of existing

barriers to the rapid and accurate

diagnosis of TB. Firstly, people who

have TB can be missed, and, for

various reasons, they do not bother

to find out the results, but return

to the community to spread the

infection. Secondly, people who

do not have TB are misdiagnosed,

because of a lack of faith in nega-

tive smear microscopy results.

Thus, scarce health system

resources are “wasted”. Thirdly,

some patients who are already

cured will be treated again since

there is no reliable way to track

the progress of the infection.

Threshold for visibility of AFB by smear microscopy

Num

ber o

f TB

baci

lli p

er m

illili

tre

(ml)

of s

putu

m

10,000

Cough worsens:patient returns

to clinic

Blood appearsin sputum;

infant daughterinfectedwith TB

Too weakto work

AFB+: TB diagnosis made

Patientvisits clinic:

no diagnosismade

First smear:AFB negative

Patientreturns

to clinic

Patient visitspharmacy

Night coughbegins

Patient feelsunwell

first month second month third month fourth month fifth month

Infection ofhealthy patient

AFB = acid-fast bacilli = smear+

Fig

ur

e 6 A TB PATIENT’S JOURNEY FROM SYMPTOMS TO DIAGNOSIS

Direct costs Indirect costs

2,5 billion

0,5 billion

Fig

ur

e 5 TOTAL ECONOMIC IMPACT OF TB IN INDIA, 1999 (US$)

Sou

rce:

refe

ren

ce 3

0.

WHO. Diagnostics for Tuberculosis: Global Demand and Market Potential. Geneva: WHO 2006.

Fluorescence Microscopy: Mercury Vapor Lamp

WHO. Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: policy statement. Geneva: WHO 2011.

Use Mercury Vapor as a light source

Staining of specimens with Auramine-O

Higher sensitivity than light microscopy,comparable specificity

Requires a dark room for examination

Light Emitting Diode (LED) Fluorescence Microscopy

Same (or slightly more) sensitivity

Cheaper and longer life duration of bulb (10,000 hr)

Cheaper microscopy

A dark room is not required

WHO recommended to use LED fluorescence microscope as a standard technique

WHO. Fluorescent light-emitting diode (LED) microscopy for diagnosis of tuberculosis: policy statement. Geneva: WHO 2011.

Methods Sensitivity and Specificity

Method Sensitivity (%) Specificity (%)

Light microscopy 32-94 94

Mercury vapor fluorescence microscopy

52-97 94

LED fluorescence microscopy 58-97 95

Steingart KR, Ng V, Henry M, et al. Lancet Infect Dis. 2006






M. tuberculosis

detection

Mycobacterial Culture

Minion J, et al. The Lancet Infectious Disease. 2010; 10 (10): 688-698.Richter E, et al. Exper Rev Resp Med. 2009; 3 (5): 497-510.

Conventional TB culture

system

Rapid colorimetric drug susceptibility test

20-30 days

Liquid culture-based technique

Mycobacterial growth indicator tube (MGIT)

7-10 days






M. tuberculosis

detection

Gene Xpert MTB/RIF: Features

Bacterial lysis

Nucleic acid extraction

Amlification

Amplicon detection

Integrated sample processing and PCR in a disposable plastic cartridge

All automatic

Boehme CC, Nabeta P, Hillermann D, et al. N Engl J Med 2010; 363:1005-1015.

Gene Xpert MTB/RIF


T h e n e w e ngl a nd j o u r na l o f m e dic i n e

n engl j med 363;11 nejm.org september 9, 20101010

97.6%. The sensitivity was 99.8% for smear- and culture-positive cases and 90.2% for smear-nega-tive, culture-positive cases, with no significant vari-ation in overall sensitivity across sites (P = 0.24 by chi-square test) (Table 2). Testing of multiple spec-imens per patient had a modest effect over the yield of a single assay performed directly on spu-tum. The sensitivity of a single direct MTB/RIF test for culture-confirmed tuberculosis was 92.2% and rose to 96.0% with the additional testing of a pel-leted sample. For the detection of smear-negative, culture-positive tuberculosis, the sensitivity of the assay was 72.5% for one test, 85.1% for two tests,

and 90.2% for three tests. A single, direct MTB/RIF test identified a greater proportion of culture-positive patients than did a single Löwenstein–Jensen culture (Table 1 in the Supplementary Appendix). Among HIV-positive patients with pul-monary tuberculosis, the sensitivity of the MTB/RIF test was 93.9%, as compared with 98.4% in HIV-negative patients (P = 0.02). There was no sig-nificant difference in sensitivity between tests on untreated sputum and those on decontaminated pellet (P = 0.16).

The estimated specificity was 99.2% for a single direct MTB/RIF test, 98.6% for two MTB/RIF tests,

Figure 2. Assay Procedure for the MTB/RIF Test.

Two volumes of sample treatment reagent are added to each volume of sputum. The mixture is shaken, incubated at room temperature for 15 minutes, and shaken again. Next, a sample of 2 to 3 ml is transferred to the test cartridge, which is then loaded into the instru-ment. All subsequent steps occur automatically. The user is provided with a printable test result, such as “MTB detected; RIF resistance not detected.” PCR denotes polymerase chain reaction.

The New England Journal of Medicine Downloaded from nejm.org by SANTI SILAIRATANA on September 7, 2014. For personal use only. No other uses without permission.

Copyright © 2010 Massachusetts Medical Society. All rights reserved.

Gene Xpert MTB/RIF: Performance


Smear-positive specimens

Smear-negative specimens

98.2% 72.5%

Sensitivitycompared with culture

99.2%

Specificity

99%Specificity

Sensitivity 98%

MTB detection

Rifampin resistance detection

Gene Xpert MTB/RIF: Pros and Cons

Adventages

Easy preparation and processing

Almost all steps run automatically

Test results can be reported within 2 hours

Can be used both for TB identificationand Rifampin susceptibility test

Disadventages

High cost

High maintenance cost

Rifampin resistance detection only

Line Probe Assay (LPA)

Rapid molecular drug resistance detection

Reverse line blot hybridization

!INNO-LiPA Rif.TB Test

Hain test: MDRTBplus, MDRTBsl


Line Probe Assay (LPA): MDRTBplus and MDRTBsl

First-line drugs Second-line drugs


Line Probe Assay (LPA)

Sensitivity Specificity

≥97% ≥99%

for detection of rifampin resistance

Sensitivity Specificity

≥90% ≥99%

for detection of combined INH-RIF resistance


LPA vs Conventional DST

Parsons LM, Somoskövi A, Gutierrez C et al. Clin Microbiol Rev. 24 (2). 2011; 314-350.

Line Probe Assay (LPA): Pros and Cons

Adventages

Rapid processing and reporting (2-7 days)

Drug susceptibility testing to INH and RIF(INNO-LiPA Rif.TB and MTBDRplus)

Drug susceptibility testing to second-lineagents (MTBDRsl)

NTM species identification

Disadventages

Labour intensive

Requires highly trained personnel

Requires dedicated laboratory space and equipment

Expensive (but cheaper than Xpert)

Indications for Rapid Drug Susceptibility Test

Risk factor(s) to carry drug resistant strains

Tuberculosis in the setting of close contact to MDR-TB patient

Positive smear at 3 months after treatment

Positive smear at 5 months after treatment

Before changing regimen or adding any drugs to the treatment regimen

Suspected NTM infection in smear positive patient






M. tuberculosis

detection

The Mantoux Tuberculin Skin Test

CDC. MMWR 2005; 54 (RR-17). American Thoracic Society and CDC. Am J Respir Crit Care Med. 2000; 161.

Injecting 0.1 mL of tuberculin purified protein derivative (PPD) into the inner surface of the forearm (intradermal injection)

Injection should be made with a tuberculin syringe

The needle bevel facing upward

The injection should produce a pale elevation of the skin 6-10 mm in diameter

Tuberculin Skin Test: Reading and Interpretation

CDC. MMWR 2005; 54 (RR-17). American Thoracic Society and CDC. Am J Respir Crit Care Med. 2000; 161.

An induration of ≥5 mm !HIV infected persons A recent contact Persons with fibrotic changes on chest radiograph consistent with prior TB Patients with organ transplants Immunosuppressed patients (e.g., >15 mg/day of prednisolone for ≥1 mo)

An induration of ≥10 mm !Recent immigrants (<5 years) from high prevalence countries Injection drug users Residents and employees of high-risk congregate setting Mycobacteriology laboratory personnel Patient with clinical conditions that place them at high risk Children <4 years of age

POSITIVEan induration

≥15 mm 48-72 hr after injection

Interferon-Gamma Release Assays (IGRAs)

QuantiFERON-TB Gold in-Tube T SPOT.TB

Measurement of a person’s immune reactivity to M. tuberculosis

Do NOT help differentiate latent tuberculosis (LTBI) from tuberculosis disease

Routine testing with IGRA is NOT recommended

Centers for Disease Control and Prevention. MMWR 2010; 59 (No.RR-5).

Characteristics of Commercially Available IGRAs

CS227840_G

National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention Division of Tuberculosis Elimination

(Page 1 of 3)

TB EliminationInterferon-Gamma Release Assays (IGRAs) – Blood Tests for TB Infection

What are they?Interferon-Gamma Release Assays (IGRAs) are whole-blood tests that can aid in diagnosing Mycobacterium tuberculosis infection. They do not help differentiate latent tuberculosis infection (LT BI) from tuberculosis disease. Two IGRAs that have been approved by the U.S. Food and Drug Administration (FDA) are commercially available in the U.S. They are:

QuantiFERON® – TB Gold In-Tube test (QFT–GIT); SPOT® TB test (T–Spot)

How do they work?IGRAs measure a person’s immune reactivity to M. tuberculosis. White blood cells from most persons that have been infected with M. tuberculosis will release interferon-gamma (IFN-g) when mixed with antigens (substances that can produce an immune response) derived from M. tuberculosis.

To conduct the tests, fresh blood samples are mixed with antigens and controls. The antigens, testing methods, and interpretation criteria for IGRAs differ (see Table 1).

What are the advantages of IGRAs? Requires a single patient visit to conduct

the test.

Results can be available within 24 hours.

Does not boost responses measured by subsequent tests.

Prior BCG (bacille Calmette-Guérin) vaccination does not cause a false-positive IGRA test result.

What are the disadvantages and limitations of IGRAs?

Blood samples must be processed within 8-30 hours after collection while white blood cells are still viable.

Errors in collecting or transporting blood specimens or in running and interpreting the assay can decrease the accuracy of IGRAs.

Limited data on the use of IGRAs to predict who will progress to TB disease in the future.

Table1: Differences in Currently Available IGRAs

QFT–GIT T–Spot

Initial Process Process whole blood within 16 hours Process peripheral blood mononuclear cells (PBMCs) within 8 hours, or if T-Cell Xtend® is used, within 30 hours.

M. tuberculosis Antigen Single mixture of synthetic peptides representing ESAT-6, CFP-10 and TB7.7

Separate mixtures of synthetic peptides representing ESAT–6 and CFP-10

Measurement IFN-g concentration Number of IFN-g producing cells (spots)

Possible Results Positive, negative, indeterminate Positive, negative, indeterminate, borderline

Centers for Disease Control and Prevention. MMWR 2010; 59 (No.RR-5).

Summary: Diagnosis of Tuberculosis

Clinical Features

Microscopy (AFB Stain)

Microbiology (Culture)

Drug susceptibility test

Imaging

Fluorescenemicroscopy

Mercury vapor LED

Liquid-based culture

MGIT

Gene Xpert MTB/RIF

Gene Xpert MTB/RIF

Line probe assays

INNO-LiPA Rif.TB MDRTBplus

MDRTBsl

Immuno reactivity test

Tuberculin skin testing

!QuantiFERON

T-spot.TB

Thank You

Health & Medicine

Tuberculosis diagnostics