MycobacteriologyReview

Margie Morgan, PhD, D(ABMM)

PPD (Purified Protein Derivative)aka the TB skin test Determined exposure to TB for decades Detects past or current TB exposure X-reacts with BCG immunization @ 25% false negative reactions Measures delayed hypersensitivity response to TB Ags r

T cells react to TB related antigens--- Lymphokine is released ---- Forms area of induration at the injection site

Measure induration in mm at 48 hr >=15 mm positive rxn or check for BCG immunization in past >=10 mm positive in immune suppressed or just exposed

Cell Mitogen assays for TB screening-IGRAs (Interferon Gamma Release Assays) QuantiFERON-TB Gold (QFT) and T-Spot Whole blood tests to screen for cell mediated immunity

to TB complex antigens Able to detect both latent TB and active infection Specific for TB complex - Useful for screening BCG

immunized population where PPD falsely positive Useful if patient cannot return to have PPD reaction

interpreted Tests quantitate the immune response to TB following

the stimulation of the patient’s T cells by TB specific antigens

Mycobacteria –Acid Fast Bacilli (AFB) Related to the genera Nocardia, Corynebacterium, and

Rhodococcus What does the term “Acid Fast” refer to?

Once stained the rods resist decolorization with acid alcohol (HCl)

Very beaded and faded on Gram stain Gram stain is NOT a good stain to detect AFB

Note: Differentiate AFB staining of the mycobacteria from partial acid fast (PAF) staining used for the Nocardia species. AFB acid fast stains use HCl to decolorize PAF acid fast stains use H2SO4 to decolorize

This is a more gentle process and Nocardia will be PAF + but will stain negative in the “true” AFB stains meant for the mycobacteria.

Gram

AFB

Mycobacteria – General Characteristics Aerobic, no spores, slightly curved

or straight rods, rarely branch, vary in length depending on species

Hardy in the environment for months Most species grow on simple substrates Mycobacteria include obligate pathogens,

opportunists and saprophytic species High amount of complex mycolic acids and free

lipids in cell wall which give many properties to this genus including AFB staining properties

Identification of the Mycobacteria For decades the identification algorithm started with

the determination of pigment in the light and dark followed by biochemical reactions

With expanding taxonomy, biochemical reactions were not able to separate and identify most of the newly recognized species so we evolved into HPLC methods. HPLC is now obsolete.

Current methods for identification: Genetic probes – RNA/DNA hybridization probes MALDI-TOF Mass Spectrometry to analyze proteins Sequencing 16 sRNA for genetic sequence information

Mycobacteria Taxonomy TB complex:

Mycobacterium tuberculosis M. bovis and the Bacillus Calmette-Guerin strain

(BCG) M. africanum And some very rare species:

Mycobacterium microti Mycobacterium canetti Mycobacterium caprae Mycobacterium pinnipedii Mycobacterium suricattae Mycobacterium mungi

Mycobacteria other than TB – “MOTT”

Mycobacteria that cause disease? Mycobacterium tuberculosis – the major pathogen of this genus

The others: M. avium-intracullare complex M. genavense M. haemophilum M. kansasii M. malmoense M. marinum M. simiae M. szulgai M. ulcerans M. xenopi M. fortuitum group M. abscessus M. chelonae M. mucogenicum

Mycobacteria that rarely if ever cause disease! If so, in immune compromised! Mycobacterium gordonae M. gastri M. celatum M. scrofulaceum M. terrae complex M. smegmatis

Algorithm for identification of MOTT- historically speaking The Runyon System is used to classify those species

not in the TB complex (MOTT) Divided into four groups:

Photochromogen = Pigment when exposed to light in Light Test

Scotochromogen =Pigment in both light and dark in the Light Test

Non-photochromogen = No pigment produced in light or dark in the Light Test

Rapid Grower = Growth rate (<= 7 days)

Light Test – does it produce a yellow pigment after being exposed to light ?

Group I PhotochromogenTurn yellow after light exposure

Group IIINon-photochromogen – No pigment produced

Group II ScotochromogenAlways has yellow pigment

Runyon Classification System Results Group I - Photochromogen – turns yellow when

exposed to light, no color in the dark M. kansasii M. simiae M. szulgai when incubated at 25˚C*** M. marinum

Group II - Scotochromogen – yellow pigment in dark or exposure to light M. gordonae M. scrofulaceum M. szulgai when incubated at 37°C***

Runyon Classification cont’d

Group III – Non-photochromogen – No pigment produced in the light or dark M. avium-intracellulare M. haemophilum

Group IV – Rapid growers – grow in 7 days or less M. fortuitum group M. abscessus M. chelonae M. mucogenicum

Specimen Processingin the AFB Laboratory

Level 3 biosafety precautions required in AFB laboratories that process, identify and perform susceptibility testing

Level 2 Hepa filter approved biosafety cabinet with return air vented to outside of the laboratory Safety cabinets must be certified at least yearly for safe use

Negative air flow, anteroom for dressing and washing hands 95 respirator masks or PAPR (powered air purifying respiratory

mask), gloves, disposable gowns must be worn Plastic cups with protected lids for centrifugation of specimens

Diagnosis: Specimen collection Sputum – 3 early morning collection or 3 specimens at least 8 hours apart Bronchial lavage fluid Tissues or Lesions CSF or sterile body fluids Urine – 3 to 5 early morning collection Stool – M. avium-intraculluare complex Gastric – children, must neutralize pH (7) of specimen after collection for AFB to survive Blood – for detection of disseminated disease

Automated systems – AFB blood culture bottles manufactured for AFB isolation

Specimen ProcessingStart to Finish! 5 ml of specimen pipetted into conical tube Decontaminate and Liquify specimen for 15 minutes

Add 5 ml of 4% NaOH (Increases the pH to 9) plus N-acetyl-L-Cysteine (breaks up the mucus)

Fill tube with phosphate buffer to neutralize pH (7.0) Centrifuge for 30 minutes

3000 X g to pellet the specimen Pour off the supernatant Prepare slides from pellet for AFB staining Dilute the pellet with small amount of sterile saline

for culture Incubate cultures @ 37˚C, 5-10% CO2 for 6–8 wks

Why do you Decontaminate Specimens? Mycobacteria are more resistant to killing by acids and

alkaline solutions than most bacteria due to the high amount of complex lipids in the cell wall – this is used to rid the specimen of contaminating bacteria and yeast

For the slow growing mycobacteria to be cultured Must eliminate competing bacteria that grow 24 x faster Must also release the AFB from mucus plugs in the sputum

specimens Accomplished by exposing the sputum to alkaline/acid solutions

and mucolytic reagents such as 4% NaOH and L-acetyl-L cysteine

Specimen Decontamination/Digestion Most often used :

4% NaOH – for decontamination N-acetyl-L-cysteine – liquid faction of mucus Expose specimen to solution for 15 minutes

Used in Special circumstances: Oxalic acid can be used for sputum collected from

patients with cystic fibrosis to kill mucoid Pseudomonas aeruginosa Oxalic acid should not be used routinely Will decrease isolation of AFB in culture

These solutions kill bacteria and can also kill AFB if left on specimen > 15 minutes

Specimen Centrifugation Centrifugation at 3000 X g (fast) Speed of centrifugation is important - AFB are lipid

laden and they will float if not spun fast enough – must pellet to bottom of tube so AFB are not poured off into waste

Helps to determine the sensitivity of the AFB stain Pour off supernatant into waste Use pellet for stains/culture

Plating -Plate and Tubed Culture Media

Middlebrook – Synthetic media Clear solid agar and liquid media Synthetic = chemical ingredients added for optimal growth Used for culture and susceptibility testing Can Autoclave for sterility

Lowenstein-Jensen – Egg based Green due to malachite green Hens egg, glycerol, and potato flour Sterilize by inspissation – drying

Cultures on incubated at 37˚C , 5-10% C0₂ for 6-8 weeks

Automated Detection of AFBBactec MGIT 960, Bacti-Alert and VersaTREKUse Liquid Middlebrook 7H9 tubed media for growthBactec and Bacti-Alert have same detection method

As AFB grow in the tubes, the AFB respire CO₂ and the O₂ is decreased. The lower level of O₂ leads to fluorescence of the tube indicator and indicates growth in the tube.

Incubation at 37˚C for 6 weeks

BACTEC MGIT 960 NAP test – Identification for TBNAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)

Automated test on NGIT 960 for TB identificationTB does not grow in the NAP containing tubeOther Mycobacteria species grow in NAP

MGIT 960BACTEC

Bacti-Alert

VersaTrek

Acid Fast Staining for Mycobacteria -only concentrated specimen should be stained Carbol Fuchsin (CF) based stains

Carbol Fuchsin is red colored stain Potassium permanganate provides background blue counterstain

Two methods: Ziehl-Neelsen (ZN) – uses heat to drive stain into lopid laden AFB Kinyoun – uses increased % of phenol to drive stain into AFB

Read numerous microscopic fields (5 min)using light microscope/100x oil objective

Fluorochrome based stain

Auramine Rhodamine – fluorescent stain, organisms stain fluorescent

yellowish with black background, Read on 25X or 40X for 2 min, viewing numerous

fields using a fluorescence microscope Nonspecific Fluorochromes that bind to mycolic

acidsConsidered more sensitive than ZN or Kinyoun stains for patient slide examination

Acid Fast staining of the Mycobacteria

Mycobacterium avium complexOrganisms are routinely shorter than TB(Kinyoun stain)

M. Tuberculosis - Organisms are long rods and can appear as if they are sticking together [cord factor]

In broth cultures -ropes of AFBwill form

Direct Detection of TB from Respiratory Specimens by Amplification Two tests are FDA cleared to detect TB complex organisms

in smear positive and smear negative sputum specimens: Hologics Amplified MTD Test - Transcription Mediated Amplification

(TMA) Cepheid Xpert-TB RIF (rtPCR) – detects TB complex and Rifampin

resistance (rpoB gene) Amplify a 16S rRNA gene sequence of TB Sensitivity of assays @ 90% AFB smear (+) specimens and

75% (+) for AFB smear (-) specimens Test of diagnosis not cure

Residual rRNA and DNA can be present up to 6m after diagnosis

Must confirm with AFB culture and sensitivity

Mycobacterium tuberculosis Optimal Temp 37˚ C, Grows in 12 –25 days Buff colored, dry cauliflower-like colony Manual tests used for identification

Niacin accumulation Positive – Niacin produced from growth of TB on egg containing medium (LJ medium) Nitrate reduction Positive– Reduces nitrate to nitrite Is it reallly M. tuberculosis or could it be M. bovis?

Both in TB complex and diseases are similar M. bovis = nitrate reduction negative M. bovis does not grow in Thiophene-2-carboxylic hydrazide (T2H),

however TB does grow on this substrate Currently ****Molecular probe, MALDI-TOF, & Sequencing

Mycobacterium tuberculosis

Demonstrates Cord factor – due to high lipid content in cell wall, rods stick together and develop long ropes – unique to M. tuberculosis

Long AFB – stick together

Tuberculosis Classic presentation

Slowly progressive pulmonary infection cough, weight loss, and low grade fever

Dissemination occurs most often in AIDS, elderly, children and with some medications

Initial focus: lesion with calcified focus of infection and an associated lymph node known as Ghon’s complex

Secondary tuberculosis: seen mostly in adults as a reactivation of previous infection (or reinfection), Granulomatous inflammation much more florid and widespread. Upper lung lobes are most affected, and cavitation can occur.

TB is spread by respiratory droplets All patients with a positive concentrated smear require respiratory

isolation precautions/ suspect for transmission All patients with high level of suspicion require precautions

TB in HIV/AIDS patients Worldwide -TB is the most common opportunistic infection

affecting HIV/AIDS patients AIDS patients have high likelihood to be muti-drug resistant

TB (MDRTB) With progressive decline of cell mediated immunity (low

CD4 count) – greater risk of extra pulmonary dissemination Granulomas with/without caseation can occur

Miliary TB

M. tuberculosis outside lung Scrofula -Unilateral lymphadenitis (cervical lymph node)

most often seen in immune compromised (50%) Fine needle aspiration for specimen

M. tuberculosis (adults) M. avium complex and other MOTT (2-10%)

most common in children

Pott’s disease - TB infection of the spine Secondary to an extraspinal source of infection. Manifests as a combination of osteomyelitis and arthritis that

usually involves more than 1 vertebra.

Susceptibility testing of TB Two methods for testing

(1) Agar dilution (indirect proportion method) antibiotics embedded in solid agar and TB plated onto media

(2) Bactec liquid 7H9 medium with antibiotic solutions Tested on the automated MGIT 960 system

Primary TB drug panel / 5 drugs Isoniazid * Ethambutol* Pyrazinamide* Streptomycin Rifampin *

PCR Methods– Hybridization probes used in combination with RT PCR assay to quantify target DNA. Used for rapid determination of MDRTB (multi drug resistance)

Mycobacterium bovis M. bovis produces disease in cattle and other animals

Spread to humans by raw milk ingestion Disease in humans similar to that caused by TB

M. bovis can be the cause of bladder infections in patients treated with BCG [Bacille Calmette-Guerin] when used as an immune adjuvant to treat bladder cancer BCG is an attenuated strain of M. bovis It can become “active” and infect the bladder M. bovis will be isolated from urine specimens

Is it TB – or is it M Bovis? M. bovis = nitrate negative, M. TB = nitrate positive M. bovis does not grow in Thiophene-2-carboxylic hydrazide

TB grows in the T2H compound M. bovis does not produce Pyrazinamidase (PYRZ) enzyme

TB produces PYRZ enzyme

Mycobacterium ulcerans Infection begins as boil and develops a painless skin

ulcer known as Bairnsdale or Buruli ulcer Can progress into avascular coagulation necrosis Found primarily in the African continent and tropical

environments Optimum growth temp 30˚ C

All skin lesions should be cultured at both 30˚ and 37˚C

Slow growing 3- 4 weeks Negative – niacin accumulation

and nitrate test

Mycobacterium kansasii Culture 37* C, 10-20 days Photochromogen Niacin accumulation test = negative Nitrate reduction = positive Tween 80 positive / tests for presence of lipase enzyme 68*C catalase positive Acid fast stain: cells are long, rectangular and beaded,

larger than TB/ Shepherd’s crook Clinical disease in adult mimics pulmonary TB but does

not disseminate from lung – predisposition diseased lung Causes granulomatous inflammation in lung

Mycobacterium marinum Optimum temp for growth is 30˚ C

Growth in 5-14 days Photochromogen M. marinum found in both fresh and salt water sources

Associated with trauma in Swimming pools, fish tanks, water cooling towers

Disease known as “Swimming pool granuloma”

Mycobacterium marinum Disease Tender, red or blue/red subcutaneous nodules following

trauma to skin Biopsy (skin) specimens must be cultured at 30*C Lesions can continue to extend up arm and spread along

lymphatics, Clinically appears similar to infections with Sporothrix,

Nocardia and rapid growing Mycobacteria species.

Mycobacterium szulgai Grows at 37 ˚C in 12 - 25 days Scotochromogen at 37˚C / Photochromogen at 25˚C

Only AFB that has a different light test result based on temperature

Niacin accumulation = negative Nitrate test = positive Unusual cause of pulmonary

disease in adultsSymptoms similar to TB

25˚ C - Photochromogen

37˚ C - Scotochromogen

Mycobacterium xenopi

Optimum temp 42˚C, capable of growing in hot water systems

Grows in 14 - 28 days in culture Scotochromogen Egg nest type colony on agar plate Rare cause of pulmonary disease

Could be considered “contaminate” Clinically disease is like TB Occurs in patients with preexisting lung lung

disease Can be seen in HIV/AIDS patients

M. avium-intracellulare complex M avium and M intracellulare – two species

Biochemically and genetically very difficult to distinguish Opportunistic infection in HIV/AIDS

High organism load in AFB stains of in intestine, liver, spleen and bone marrow

Isolated from blood cultures Involvement of GI tract can cause diarrhea

Positive AFB smears in stool Tissue Biopsy with inflammation

Organisms mostly short rods Shorter than TB organisms Do not have cord factor

Pathology - Necrotizing rather than granulomatous inflammation

M avium-complex in lymph node tissue – Kinyoun AFB stain

M. avium complex Kinyoun AFB stain variable in size – absence of cording

Granulomatous inflammationIn lung tissue – H&E stain

Bowel -Lamina propria expanded from predominately Lymphohistocytic infiltration

M. avium-intracellulare

Laboratory – Growth at 37 ˚C / 7 – 21 days Non-photochromogen Smooth colony Inert in biochemicals Identify using

GenProbe (AccuProbe) molecular identification MALDI-TOF Genetic 16s rRNA Sequencing

M. avium complex clinical correlation HIV/AIDS

Disseminated infection in end stage AIDS disease Nonspecific low grade fever, weakness, weight loss,

picture of fever of unknown origin Abdominal pain and/or diarrhea with malabsorption

Normal host In adults pulmonary disease, much like TB,

marked % of cases – elderly adults with lung damage (history of smoking)

In children – lymphadenitis M. avium ssp. paratuberculosis –

? Association with Crohn’s disease Inconclusive evidence for causation

Rapid growing Mycobacteria species Laboratory

Growth at 37˚C in <=7 days Positive Arylsulfatase test @ 20 species / most common:

M. fortuitum – skin and surgical wound infections M. chelonae- skin infections in immune suppressed M. abscessus - chronic lung infection and skin infection in

immune suppressed Biochemical reactions:

M. fortuitum Nitrate Positive and Iron Uptake positive M. chelonae and M. abscessus Nitrate Negative

MALDI-TOF and Sequencing for identification

Miscellaneous

M. gordonae – Rare! Cause of Infection Major laboratory water contaminant in cultures

”tap water bacillus” – present in water systems Use sterile water in culture processing to prevent

contamination Scotochromogen

Miscellaneous Mycobacterium haemophilum

Requires addition of hemoglobin or hemin to culture media for organism growth Will not grow on LJ or in automated systems without the

addition of hemin supplements Painful subcutaneous nodules and ulcers,

primarily in AIDS patients or immune suppressed Lymphadenitis in children

Mycobacterium leprae Leprosy – also known as Hansen’s disease Begins with anesthetic skin lesions and peripheral neuropathy with nerve thickening Presenting presentation numbness in earlobes or nose Lapromatous leprosy – severe disfiguring lesions, large

numbers of AFB in lesions / associataed with co-infection with Strongyloides stercoralis

Tuberculoid leprosy -less severe/fewer lesions, lower numbers of AFB in lesions

Will not grow on laboratory AFB media Armadillo is the natural reservoir

PCR on tissue for definitive diagnosis

Tuberculoid leprosy

Lapromatous leprosy

Skin biopsy - AFB seen in nerve fiber

Organisms in“cigar packets”