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

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

TECHNIQUES

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

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)

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+

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

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

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

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

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

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

Classifying the tuberculin reaction

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

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

Newer techniques

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

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

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

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

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)

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?

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

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

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

Chromatography

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

studies• Results within 2 hours• High cost

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).

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

Available Interferon-gamma release assays (IGRA) tests

Quantiferon Gold T-SPOT.TB

Limitation is COST. NRs 4000/-

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

Disadvantages of IGRA

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

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

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

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

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

FAST-Plaque-Response

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

Adenosine Deaminase (ADA)

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

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

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

Dr.T.V.Rao MD 51

Thank you