tuberculosis diagnostics
DESCRIPTION
Information about diagnostic methods and techniques for tuberculosis including microscopy, fluorescence microscopy, mycobacterial culture, molecular techniques (line probe assay, Xpert MTB/RIF), interferon gamma release assay (IGRA) and tuberculin skin test (TST)TRANSCRIPT
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
Immune reactivity detection
Detecting TB Infection
Microscopic examination
Gene/molecular- based techniques
Mycobacterial culture
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
Immune reactivity detection
Detecting TB Infection
Microscopic examination
Gene/molecular- based techniques
Mycobacterial culture
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
Boehme CC, Nabeta P, Hillermann D, et al. N Engl J Med 2010; 363:1005-1015.
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
Boehme CC, Nabeta P, Hillermann D, et al. N Engl J Med 2010; 363:1005-1015.
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
O’Grady J, Maeurer M, Mwaba P et al. Current Opinion in Pulmonary Medicine 2011, 17; 134-141.
Line Probe Assay (LPA): MDRTBplus and MDRTBsl
First-line drugs Second-line drugs
O’Grady J, Maeurer M, Mwaba P et al. Current Opinion in Pulmonary Medicine 2011, 17; 134-141.
Line Probe Assay (LPA)
Sensitivity Specificity
≥97% ≥99%
for detection of rifampin resistance
Sensitivity Specificity
≥90% ≥99%
for detection of combined INH-RIF resistance
O’Grady J, Maeurer M, Mwaba P et al. Current Opinion in Pulmonary Medicine 2011, 17; 134-141.
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
Immune reactivity detection
Detecting TB Infection
Microscopic examination
Gene/molecular- based techniques
Mycobacterial culture
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