MycobacteriologyReview Update 2017

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

T cells react to TB and related antigens Lymphokine is released Forms area of induration at the injection site Measure area of 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 is 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 Ags Sensitivity is somewhat equal to PPD but better specificity What is a “TRUE” positive reaction is the issue! A fluctuating

immune response can ride the positive cut off value.

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

Survive 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 acid fast 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 High Performance Liquid Chromatography (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: MOTT 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 Runyon System

Used to classify those species not in the TB complex (MOTT) Based on pigment production when exposed to light & growth rate

Runyon separates MOTT into four groups using the light test to promote pigment production Photochromogen = Pigment produced with light Scotochromogen = Pigment in both light and dark Non-photochromogen = No pigment produced Rapid Grower = Growth rate (<= 7 days)

Light Test – does it produce a yellow pigment after being exposed to 8 hours of light bulb.

Group I PhotochromogenTurns yellow after light exposure

Group IIINon-photochromogen – No pigment produced after light exposure

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 M. xenopi

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

Safety in 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, contiguous autoclave 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

Biosafety Cabinet

PAPR

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 – for M. avium-intracellulare 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 – safety cups

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? 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

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

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 for sputum collected from patients with

cystic fibrosis -eliminate mucoid strains of 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) with safety cups in

place 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 with supernatant

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

Manual Plating/ Culture Media

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

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

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

Automated Detection of AFBBactec MGIT 960, Bacti-Alert and VersaTREKLiquid 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 Fluoresce

NAP test – Identification for TB complexNAP = (p-nitro-α-acetylamino-B-hydroxypropiophenone)

Automated test run only on the MGIT 960 instrumentTB complex does not grow in the tube containing NAP MOTT species grow in NAP

MGIT 960BACTEC

Bacti-Alert

VersaTrek

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

Carbol Fuchsin is a red colored stain Potassium permanganate is the background blue counterstain

Two methods: Ziehl-Neelsen (ZN) – uses heat to drive stain into lipid 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 stainAuramine Rhodamine –

fluorescent stain, organisms stain fluorescent yellow with black background,

Read on 25X or 40X for 2 min, viewing numerous fields using a fluorescence microscope

Based on nonspecific fluorochrome that binds to mycolic acids

Considered more sensitive than ZN or Kinyoun stains for concentrated specimen patient slide examination

Acid Fast staining of the Mycobacteria

Mycobacterium avium complexOrganisms are routinely shorter than TB(Kinyoun stain) No cording

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 Molecular Amplification Two tests FDA cleared to detect TB complex organisms in

smear positive and smear negative sputum specimens: Hologic Amplified MTD Test - Transcription Mediated Amplification Cepheid Xpert-TB RIF (rtPCR) – detects TB complex and Rifampin

resistance (rpoB gene)

Amplify a 16S rRNA gene sequence of TB complex 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 a positive diagnostic test

Must confirm all specimens with AFB culture and sensitivity

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

Niacin accumulation Positive Niacin produced from growth of TB on egg containing

medium (LJ medium) Nitrate reduction Positive

Is it really 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 Current ID uses 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 Disease

Slowly progressive pulmonary infection cough, weight loss, and low grade fever Lesion with calcified foci of infection and an associated lymph node known as

Ghon’s complex Dissemination occurs most often in AIDS, elderly, children and with

some medications (Remicade- infliximab) Secondary tuberculosis: mostly in adults as 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 All patients with high level of suspicion require precautions

Pathology of M. tuberculosisLung with pulmonary TB has apical cavitations with numerous areas of caseous necrosis and massive tissue consolidation

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

affecting HIV/AIDS patients AIDS patients have higher likelihood to be multi-drug

resistant (resistant to at least INH and Rifampin) 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 diagnostic specimen

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

most common in children

Pott’s disease - TB infection of the spine Secondary to an extra-spinal 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 inoculated onto media

(2) Bactec liquid 7H9 medium containing antibiotic solutions Tested on 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 is used by public health labs for rapid determination of MDRTB

Mycobacterium bovis Produces disease in cattle and other animals

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

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 M bovis or is it M tb?M.bovis M. tb

Nitrate Negative PositiveGrowth in T2H* No growth GrowthPyrazinamidase Negative Positive

*(Thiophene-2-carboxylic hydrazide)

Mycobacterium ulcerans Bairnsdale or Buruli ulcer boil that progresses into a

painless skin ulcer Can progress into avascular coagulation necrosis African continent and tropical environments

Optimum growth temp 30˚ C All skin lesions should be

cultured at both 30˚ and 37˚C Slow growing requiring 3- 4 weeks

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

AFB are larger than TB - rectangular and beaded Shepherd’s crook shape

Clinical disease mimics pulmonary TB but usually does not disseminate from lung – Predisposition diseased lung (COPD) Immune suppressed, pneumoconiosis, alcohol abuse, HIV Granulomatous inflammation in lung

Mycobacterium marinum Optimum temp for is 30˚ C

Grows poorly or not at all at 37°C Grows in 5-14 days Photochromogen

M. marinum found in fresh and salt water Associated with trauma occurring in water:

swimming pools (swimming pool granuloma) cleaning fish tanks ocean (surfing)

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

develop following trauma Biopsy (skin) specimens for culture and histopathology Lesions can spread up arm and 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 of incubation

Rare cause of pulmonary disease in adults

Symptoms 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

Clinically disease is like TB Occurs in patients with preexisting lung disease and

HIV/AIDS

M. avium-intracellulare complex M avium & M intracellulare most common but complex

includes eight species of environmental and animal related AFB

Biochemically and genetically very difficult to distinguish Growth at 37 ˚C / 7 – 21 days Non-photochromogen Smooth colony Inert in biochemicals Identify using

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

M. avium-intracellulare complex Opportunistic infection

High organism load in intestine, liver, spleen and bone marrow Can be recovered from blood culturesInvolvement of GI tract causes diarrhea

Positive AFB stool smears Tissue Biopsy with inflammation

AFB small and not beaded Do not have cord factor

Pathology - Necrotizing rather than granulomatous inflammation

M avium-complex in lymph node tissue (Kinyoun stain)

Granulomatous inflammationlung tissue (H&E stain)

Bowel -Lamina propria expanded from predominately Lymphohistocytic infiltration

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 mostly pulmonary disease, much like TB

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

M. chimaera Contaminated Heater–cooler units using tap water identified as a

source of surgical site infections. Units caused an airborne transmission of M. chimaera over an open surgical field

Rapid growing Mycobacteria species Low virulence, found in nature Cause: Infections in soft tissue, venous catheter sites

and shunts @ 20 species – 5 spp. 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 M. smegmatis M. mucogenicum

Grows in <=7 days = rapid grower Biochemical reactions:

All species are Arylsulfatase test positive M. fortuitum: Nitrate Positive and Iron Uptake positive M. chelonae and M. abscessus: Nitrate Negative

MALDI-TOF and 16 S RNA Sequencing for identification

Miscellaneous

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

because it is commonly contaminating water systems

Use sterile/distilled water in AFB culture processing to prevent contamination

Miscellaneous pathogenic species Mycobacterium haemophilum

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

addition of hemin supplements Disease: Painful subcutaneous nodules and

ulcers, primarily in AIDS patients or immune suppressed

Lymphadenitis in children

Mycobacterium leprae Leprosy – Hansen’s disease Begins with anesthetic skin lesions, peripheral neuropathy with nerve thickening,

numbness in earlobes or nose, loss of eye brows Two types of Leprosy

Lapromatous – severe disfiguring lesions, large numbers of AFB in lesions / associated with co-infection with Strongyloides stercoralis

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

Will not grow on laboratory AFB media PCR on tissue for definitive diagnosis Armadillo is the natural reservoir

Tuberculoid leprosy

Lapromatous leprosy

Skin biopsy - AFB seen in nerve fiber

AFB in “cigar packets”