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Page 1: Investigations in Tuberculosis and advances

Investigations in Mycobacterium Tuberculosis and Advances

Dr. Nirish VaidyaResident, First yearInternal Medicine

KUSMS, Kathmandu University Hospital

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Mycobacterium tuberculosis-Characteristics

•Gram positive •Obligate aerobe•Non-spore-forming•Non-motile rod•Mesophile•Slow generation time: 15-20 hours

•May contribute to virulence

•Lipid rich cell wall contains mycolic•Responsible for many of this bacterium’s characteristic properties

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The Burden• Worldwide, 9.6 million people are estimated to have fallen ill with TB in

2014• In 2014, 6 million new cases of TB were reported to WHO, fewer than

two-thirds (63%) of the 9.6 million people estimated to have fallen sick with the disease. This means that worldwide, 37% of new cases went undiagnosed or were not reported.

• Of the 480 000 cases of multidrug-resistant TB (MDR-TB) estimated to have occurred in 2014, only about a quarter of these – 123 000 – were detected and reported

• Delay in the diagnosis?• Delay in getting result?• To reduce this burden, detection and treatment gaps must be

addressed, funding gaps closed and new tools developed• Laboratory methods play a crucial

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TECHNIQUES

1. Detection of Mycobacterium2. Molecular Methods3. Identifications of Species4. Immunological Methods

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Sputum Microscopy• Carbol fuchsin • Fluorochrome stain such as auramine-rhodamine– Improves the sensitivity of Mtb detection– Recent advances in light-emitting diode (LED)

technology have widened the applicability of fluorescent microscopy

• Persons unable to cough up sputum– induce sputum– bronchoscopy – gastric aspiration The quantity of sputum (at least 5 mL)

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Sputum smears stained by Z-N stain

Advantage: - cheap – rapid - Easy to perform - High predictive value > 90%

-Specificity of 98%Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.)

Sensitivity: 40-70% - Young children, elderly & HIV infected persons

may not produce cavities & sputum containing AFB.Solution:

-Centrifused sample-Overnight Sedimentation

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New Policy and Smear microscopy definition of a TB case

• New definition in 2007 (ISTC):“person with al one AFB is sufficient out of a total of two examined”

• 2 samples regardless the collection time– Retrospective study

• Nelson, SM, Deike, MA, Cartwright, CP. Value of examining multiple sputum specimens in the diagnosis of pulmonary tuberculosis. J Clin Microbiol 1998; 36:467. – overall, 92 percent of cases would have been detected with two specimens

• Craft, DW, Jones, MC, Blanchet, CN, et al. Value of examining three acid-fact bacillus sputum smears for the removal of patients suspected of having tuberculosis from the "airborn precautions" category. J Clin Microbiol 2000; 38:4285. – a third sputum smear was of no additional value

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Interpretation of sputum stained by Z N Stain (WHO )

No No AFB/300 OIF Negative

1-9 No of AFB/100 OIF Mention the number

10-99 No of AFB/100 OIF 1+

1-10 No ofAFB/50 OIF 2+

>10 No of AFB/ 20 OIF 3+

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The smear may be stained by auramine-O dye. TB bacilli are stained yellow against dark

background & easily visualized using florescent microscope.

Advantages: - More sensitive - RapidDisadvantages: - Hazards of dye toxicity - More expensive

LED Fluorescence Microscopy

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Culture

• GOLD STANDARD• Solid media: Lowenstein-Jensen– Brown, granular colonies "buff, rough and tough").– Four weeks, due to the slow doubling

• Liquid media: used in commercially available automated systems

• Used to confirm diagnosis of TB

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Cultures• Sensitivity: 80-85%• Specificity: 98%• Times needed:

– Solid medium• 4-8 wks

– Liquid medium• 2 wks

Advantages: - More sensitive (need lower no. of bacilli 10-100 / ml) -Differentiate between TB complex & NTM using biochemical

reactions - Sensitivity tests for antituberculous drugs Disadvantages: Slowly growing ( up to 8 weeks ) in solid medium

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AFB smear vs. Cultures

• AFB smear– Need 5000-10000/ml bacilli– Rapid diagnosis

• Cultures– Need lower no.10-100/ml bacilli– More sensitive – Allows drug sensitivity test

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Screening-Tuberculin test ( Mantoux test)

• Delayed HSR against tubercular protein

• Inject intradermally 0.1 ml of 5TU PPD tuberculin

• Read reaction 48-72 hours after injection

• Measure only induration• Record reaction in mm

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Classifying the tuberculin reaction

• >5 mm is classified as positive in– HIV-positive persons– Recent contacts of TB case– Persons with fibrotic changes on CXR consistent

with old healed TB– Patients with organ transplants and other

immunosuppressed patients

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Classifying the tuberculin reaction

• >10 mm is classified as positive in– Recent arrivals from high-prevalence countries– Injection drug users– Residents and employees of high-risk settings– Mycobacteriology laboratory personnel– Persons with clinical conditions that place them at

high risk– Children <4 years, or children and adolescents

exposed to adults in high-risk categories

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Classifying the tuberculin reaction

• >15 mm is classified as positive in– Persons with no known risk factors for TB

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Tuberculin Test: Interpretation

• A positive test

• The person’s body was infected with TB bacteria• Latent TB infection or TB disease??-Additional test needed• may develop reactivation type of T.B.

• A negative tuberculin test

• excludes infection in suspected persons • The person’s body did not react to the test, and that latent

TB infection or TB disease is not likely.• are at risk of gaining new infection

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False negative

•Severe tuberculosis infection (Miliary T.B.) - Hodgkin’s disease • Corticosteroid therapy -

Malnutrition - AIDS• Children below 5 years of

age with no exposure history

False positive

• Atypical mycobacterium• BCG vaccination

– Usually <10 mm by adulthood

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Recombinant Early Secretory antigenic target (ESAT)-6 instead of tuberculin have demonstrated safety and tolerability of such a test. May solve the problem but is on Phase I trial

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Newer techniques

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Septi-check AFB• Simultaneous detection of

Mycobacterium tuberculosis and non tuberculosis mycobacterial

• Requires ~ 3 weeks of incubation• Specimens: Sputum,

bronchoalveolar lavage, urine, stool, body fluids, biopsy tissues, wounds and skin

• Advantage over convetions Mb isolation media-Small inocula-Short time

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Microcolony detection on solid media(MODS)

• Uses simple light microscopy on plates with a thin layer of 7H11 agar medium

• Identifies characteristics strings and tangles of M.tb

• Rapid yield ~9-10 days• Low cost alternate for

detecting drug resistance• Sensitivity 92%• High technical skills,

biosafety cabins complex agar medium

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Mycobacteria Growth Indicator Tube (MGIT)

Tube contains modified Middlebrook 7H9 broth base

All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood) could be cultured

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BACTEC • Specimen is inoculated in 14C labeled

7H12 medium(MGIT)• Incubated• Mycobacterium multiply generating

14Co2, increase fluorescence in sensor of the bottle

• The instrument scans the MGIT every 60 minutes for increased fluorescence.

• An instrument-positive tube contains approximately 10^5 to 10^6 CFU/mL.

• Can detect growth as early as 4 days and drug susceptibility in 21-28 days.

• Cost effective

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MB/Bac T• Non-radiometric continuous monitoring

system that utilizes colorimetric sensor and reflected light to continuously monitor the Co2 concentration in culture medium

• Good alternative to BACTEC– But takes slightly longer time ~15-20 days

• The mean time to detection was 12.9 days by BACTEC MGIT960, and 15.0 days with BACTEC 460,compared with 27.0 days LJ Medium (Advances in the diagnosis of tuberculosis; C LANGE et al;Repirology)

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ESP Blood culture system

• Fully automated continuous monitoring culture system by evaluating gas consumption by microbes rather than production of CO2 as in BACTEC

• Mean time of detection 11-16 days

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• Detection and Diagnosis• – uncultivable or difficult to culture

• – need for rapid answer• Prognosis and management• – need for quantitative

information (viral load)• – susceptibility testing (drug

resistance) without culture• • Molecular resistance

testing

Molecular DiagnosticsWhy?

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Nucleic Acid Amplification (NAA)

• Both detection and identification of M. tuberculosis through enzymatic amplification of bacterial DNA

• Amplify M. tuberculosis-specific nucleic acid sequences using a nucleic acid probe.

• Used mostly for confirmation of smear positive results

• Quick results, diagnostic accuracy• Require as few as 1O bacilli from a given sample• Expensive

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Nucleic Acid Amplification (NAA)

• Specificity in the range of 98% to 99%. • Useful technology for rapid diagnosis of smear

negative cases of active TB• Able to identify 50-60% of smear -ve culture +ve cases• Distinguish M.tb from NTM in smear +ve cases• Should not be used to replace sputum microscopy as

an initial screen & culture remains the gold standard• Very high degree of quality control required

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Polymerase Chain Reaction (PCR)

• Allows sequences of DNA to be amplified in vitro

• Detection is based on multiplication not of whole bacilli, as in culture, but of their genetic material, chromosomal DNA or ribosomal RNA, typical for MTB or MTB complex.

• Most common target used for PCR is insertion sequence IS6110 which is specific for M. tb.

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Advantages: - Rapid procedure ( 3 – 4

hours) - High sensitivity (1-10 bacilli / ml sputum)Sensitivity ~95%, Specificity

~100%Disadvantage

-High cost

Polymerase Chain Reaction (PCR)

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Xpert MTB/RIF • Real time PCR assay to amplify a specific

sequence of the rpo B gene• Xpert MTB/RIF detects M. tuberculosis as well

as rifampicin resistance• Cartridge based Automated molecular test

M.Tb and its resistance to Rifampin

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Advantages with GeneXpert

• Simultaneous detection of both MTB and rifampicin resistance, a marker for MDR strains

• Unprecedented sensitivity for detecting MTB — even in smear negative, culture positive specimens

• Results in two hours; requires no instrumentation other than the GeneXpert® System

• On-demand results enable physicians to treat rapidly and effectively

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• Monoresistance to Rifampicin ~5 %• Concurrent resistance with Isoniazide ~95%– Dx of MDR Tb with high level of accuracy

• Xpert MTB/RIF has higher sensitivity for Sputum smear positive cases than Smear negative cases. Nontheless a valuable tool for smear negative cases

• International standard for TB Care recommended Xpert MTB/RIF assay with Culture for Sputum Negative Suspected cases

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Chromatography

• Identification of different species• Used in reference labs for epidemiologic

studies• Results within 2 hours• High cost

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TB PNA FISH

• Fluorescence in situ hybridization (FISH) using specific Peptide nucleic acid (PNA) probe allows differentiation between tuberculous and nontuberculous mycobacteria in AFB+ve cultures

• Specificity and predictive values approaches 100%

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• DNA amplification technique• It is a variant of PCR, in which a

pair of oligonucleotides are made to bind to one of the DNA target strands, so that they are adjacent to each other. A second pair of oligonucleotides is designed to hybridize to the same regions on the complementary DNA.

Ligase Chain Reaction

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• The action of DNA polymerase and ligase in the presence of nucleotides results in the gap between adjacent primers being filled with appropriate nucleotides and ligation of primers

• Mainly being used for respiratory sample

• High overall specificity and sensitivity for smear +ve and –ve specimens.

Ligase Chain Reaction

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Loop-mediated isothermal amplification (LAMP)

• The mixing of all reagent in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63°C 60-min incubation time.

• It is a novel nucleic acid amplification method• Used for detection of M.tb complex, M.avium, and

M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium (Ogawa’s medium).

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Interferon-gamma release assays (IGRA)

• Measure interferon (IFN-γ) released by sensitized T cells after stimulation by M. tuberculosis antigens. Measures immune reactivity to M.tb.

• The test uses synthetic peptide antigens (ESAT-6,CFP-10) that simulate this protein to generate the immune response

• No memory as in Mantoux, but tells us it’s a RECENT INFECTION

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Available Interferon-gamma release assays (IGRA) tests

Quantiferon Gold T-SPOT.TB

Limitation is COST. NRs 4000/-

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Advantages of IGRA

• Quantitative reports– postive or negative

• Result in single patient visit• Not affected by BCG status and NTB• Not affected by repeated IGRA

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Disadvantages of IGRA

• Does not tell Latent or active TB• COST• Results altered in immune compromised

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Monocyte-amplified INF-γ release assays (MIGRAS)

• Principle:– INF release leads to subsequent release of INF-

responsive chemokines such as MIG and IP-10– Measures this chemokines

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Transcription mediated amplification (TMA)

• Can identify the presence of genetic information unique to M. tuberculosis directly from pre processed clinical specimens

• Sensitivity ~ culture• Sensitivity ~96% and Specificity 100%• Disadvantage– Positive results in both viable and dead bacilli

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Other approaches

• Detection of anti-mycobacterium superoxide dismutase antibodies

• MPB 64 patch test– Transdermal patch containing MPB 64, an antigen

specific for M tuberculosis – Results obtained d after 3-4 days– Sensitivity 97.8% and Specificity 100%– Discriminated latent infection from active disease

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FAST Plaque TB

• Specific mycobacteriophages infect M.tb in specimen

• Intracellular phages undergo replications with subsequent release of phages from host cells

• Released phages are incubated along with other nonpathogenic organisms in agar plate – produces zone of clearing (plaques) – POSITIVE

• Rapid <24 hours

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FAST-Plaque-Response

• Extention of FAST-Plaque TB• Allows early detection of Rifampicin resistance

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Adenosine Deaminase (ADA)

• Surrogate marker in pleural, pericardial and peritoneal fluids and CSF

• Sensitivity of 100%, Specificity 94.6% (study performed in India)

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Detection of lipoarabinomanan

• LAM is a cell lipopolysaccharide Ag of M tb• LAM-ELISA assay in urinary useful in HIV

associated tuberculosis with low CD4 counts• Sensitivity 93%, Specificity 95%• Useless in Blood TB-ELISA

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Dr.T.V.Rao MD 51

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Thank you


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