Atypical mycobacterium

1. Mycobacteria other than
tuberculosis (MOTT)
Dr Nisha Singh
GUIDE : S.S Raut

2.  Nontuberculous mycobacteria (NTM), also known
as environmental mycobacteria, atypical mycobacteria
and mycobacteria other than tuberculosis (MOTT), are
mycobacteria which do not cause tuberculosis or leprosy
 Mycobacterium tuberculosis complex includes M.tuberculosis
M.bovis
M. africanum
M. caprae
M. microti
M. pinnipedii
 Leprosy (M. leprae)

3. Runyoun classification NTM have been categorized into four
groups by Runyoun (1959) based on pigment production and the
growth rate
 Photochromogenes
M. kansasii, M. simiae M. marinum.
 Scotochromogens
M. scrofulaceum ,M. szulgai, M. xenopi
 Non- photochromogens
M. avium, M. intracellulare, M. ulcerans
 Rapid growers
M. fortuitum , M. chelonae, M abscessus

4. Runyon
Group
Number
Group Name Description
I Photochromogens Colonies of NTM that develop pigment
following exposure to light after being grown in
the dark and take more than 7 days to appear
on solid media
II Scotochromogens Colonies of NTM that develop pigment in the
dark or light and take more than 7 days to
appear
on solid media
III Nonphotochromogen
s
Colonies of NTM that are nonpigmented
regardless of whether they are grown in the
dark or light
and take more than 7 days to appear on solid
media
IV Rapid-growers Colonies of NTM that appear on solid media in
less than 7 days

5. Species potentially pathogenic in human
M. avium intercellulare complex
M. kansasii
M. scrofulanceum
M.xenopi
M.szulgai
M.malmoense
M.simiae
M.marium
M.ulceran
M.haemophilum
M.celatum
Rapid growers :M.fortuitum, M.chelonae,M.abscessus

6. Saprophytic mycobacterium rarely causing disease in
human
M. gordonae
M. asaticum
M terrae
M. Trivial
M.Shimoidei
M. Gastri
M.Paratuberculosis
Species with intermediate growth rate
M. flavescens
Other rapid growing species
M.thermoresistible
M.smegmatis
M.phlei

7.  They are not usually transmitted from person to person
 Source of infection is water, soil, food and animals
 Human infection with NTM is common in some areas,
disease is rare
 Exhibits dysgonic growth on LJ medium
 Niacin and nitrate reduction tests are negative
 Not able to cause progressive disease in guniea pigs

8.  Immunosuppression ( HIV, Medications )
 Aging
 BCG vaccination
 Cystic fibrosis
 Fibronodular bronchiectasis
 Number of inherited and acquired defects in the host immune
response, particularly those that affect the Th1 cell and
macrophage pathway

9.  Interferon gamma receptor deficiencies
 Signal transducer and activator of transcription 1 (STAT1)
deficiency
 Auto-antibodies to interferon gamma
 CD4 lymphopenia due to HIV or other causes
 Use of TNF-alpha inhibitors

10. No typical Manifestations
 Fever
 Weight loss
 Enlarged lymph glands
 Diarrhoea
 Sweating, excessive -- night sweats
 Fatigue
 Malaise
 Cough
 Dyspnoea
 Skin lesions
 Joint pain , Bone pain

11.  Chronic bronchopulmonary disease
M. avium intercellulare complex ,M. kansasii, M.xenopi
 Cervical or other lymphadenitis
M. avium, M. scrofulanceum, M.malmoense, M.abscessus
 Skin and soft tissue disease
M. avium, M.fortuitum, M.chelonae, M.abscessus
 Disseminated infection
M. avium, M. kansasii, M.chelonae, M.haemophilum
 Catheter-related infections
M. fortuitum, M. abscessus, M. chelonae
 Skeletal (bones, joints, tendons) disease
M. marinum, M. avium complex, M. kansasii

12. The important species in this group are
M. kansasii
M. marinum
M. simiae
M. asiaticum
M. genavense

13.  It causes chronic pulmonary disease resembling tuberculosis
 It may also occasionally cause infections of the cervical
lymph nodes, penetrating wound infections and
granulomatous synovitis
 It can produce generalized infection in HIV patients

14.  Cultured at 370 C in 10-20 days
 Photochromogen – turns yellow after light exposure
 Niacin test = negative
 Nitrate reduction = positive
 Tween 80 + tests for lipase enzyme
 680C catalase +
 Acid fast stain: cells are long, rectangular and beaded, larger
than TB/ Shepherd’s crook

16.  First isolated from fish in 1926
 Causes a warty skin lesion known as swimming pool or fish
tank granuloma
 Introduce into the skin at the site of trauma
- Most infections are of hand feet arm legs
 Closely resembles M. kansasii but can be differentiated by its
poor growth at 370C,
negative nitrates test
positive pyrazinamide hydrolase
L- fucosidase activities

18.  It is the most common species associated with human
pathology . It was first isolated from rhesus monkey in 1965
 M. simiae complex is comprised of several phylogenetically
related species, including M. simiae, M. triplex, M. genavense
 Appearing rust-colour after exposure to light
 Only NTM that, like M. tuberculosis, is niacin positive

19.  First isolated from monkeys in 1965
 Dysgonic and yellow colonies on Löwenstein-Jensen
 Slow growth on LJ medium at 37°C after 15–21 days
 Unique 16S rRNA sequence
 Rarely causes human pulmonary disease

20. The important species in this group are
M. scrofulaceum
M. flavescens
M. gordonae
M. szulgai
M. xenopi
M.celatum
They are widely distributed in the environment and sometimes
contaminate cultures of tubercle bacilli

21.  It may cause scrofula (cervical adenitis) in
children
 Colonies of M. scrofulaceum grow slowly (4-6 weeks) at
various temperatures (21°, 31°, 37° C).
 They are typically smooth, buttery in consistency, and
globoid, with pigmentation ranging from light yellow to deep
orange.

22. Scrofula (cervical adenitis)

23. M. gordonae
 They are often found in tap water (the tap water
scotochromogen)
 Common contaminant in clinical specimens
 Rare cause of pulmonary disease
.

24. M. szulgai
 An uncommon cause of pulmonary disease and bursitis
 It is scotochromogenic at 37°C but
photochromogenic at 25°C
 It is most closely related to M. malmoense ,very difficult to
distinguish phenotypically
 Nitrate test positive
 Niacine test negative

25.  Optimum temp is 42˚C so it is capable of growing in hot
water supplies
 Grows in 14 - 28 days in culture
 Egg nest colony on Middlebrook agar
 Rare cause of pulmonary infections
› clinically like TB
› mostly in patients with preexisting lung disease
› Can be seen in HIV/AIDS patients

26. Non- photochromogens
Medically important species in this group are
M. avium
M. intracellulare
M. ulcerans
M. terrae
M. trivale
M. paratuberculosis
M. shimoidae

27. M. avium and M. intracellulare are so similar that that they
have been considered as one group, the M. avium complex
(MAC)
MAC complex cause lymphadenopathy, pulmonary lesions or
disseminated disease, particularly in AIDS patients

28.  Most common NTM seen in lung disease
 It is ubiquitous in the environment
 Skin lesions are uncommon.
 Survives inside macrophage
 Can spread to blood,lungs,spleen,liver and bone marrow and
intestines in HIV patients
 MAC lymphadenitis predominantly - children aged 1-4
years(cervical lymph node)
 Cure rate 30-85%

29. Mycobacterium avium complex
Organisms are routinely shorter than TB
M. Tuberculosis - Organisms are
long and can appear as if they are
sticking together [cord factor]

30.  MAC in HIV pts is theorized to represent recent acquisition
rather than latent infection reactivating
 Risk of MAC is inversely related to the patient's CD4 count,
and increases significantly when the CD4 count < 50
cells/mm³
 Disseminated MAC (DMAC) infection usually develops in
advance AIDS (bacteria subsequently spread
hematogenously to the liver, spleen, bone marrow, and other
sites)
 Clarithromycin/azithromycin – prophylaxis (life long)

31. Lady Windermere syndrome-
 Infection in the lungs due to MAC
 It is associated with suppression of cough in otherwise
healthy, thin, elderly women
 It is named after a character in Oscar Wilde’s play Lady
Windermere's Fan
 Patients with this syndrome experience chronic cough,
shortness of breath, fatigue and other less specific symptoms

32. M. avium
• Typically causes unilateral swelling of one of the lymph nodes of neck
in children . This node is firm at the beginning, but eventually
collarstud abscess is formed
• It causes natural tuberculosis in birds and lymphadenopathy
in pigs
M. intracellulare
• Is commonly known as Battey bacillus
M. paratuberculosis –
• Association with Crohn’s disease
• Also known as Johne's bacilli

33.  It causes necrotising infection of skin and subcutaneous
tissue
 It does not live freely in the environment
 The organism occupies a specific place within aquatic
environments (e.g. small aquatic animals, biofilms) from
where it is transmitted to humans by an unknown mechanism.
 It produces a destructive toxin, mycolactone, which causes
tissue damage and inhibits the immune response

34. Buruli ulcer
 It is devastating skin disease caused by M.ulcerans
 MC in Central and West Africa
 Painless lesion, grows slowly, itchy.
 Nodule of 1-2 cm develops 7-14 days after infection through
broken skin.
 After 1-2months the nodule breaks down to form a shallow
ulcer, spreads rapidly and may involve up to 15% of skin
surface.
 Severe infections may destroy blood vessels, nerves, and
invade bone deformities
 WHO began its Global Buruli Ulcer Initiative in 1998

35. (a) nodule on abdomen of a young child
(b) non-ulcerative oedematous swelling of child's hand
(c) ulcer covering most of a young child's arm
(d) a plaque

36. Rapid growers
 This is a heterogeneous group of mycobacteria capable of
rapid growth, colonies appearing within 7 days of incubation
at 370C or 250C
 Within this group, photochromogenic, scotochromogenic,
and non-chromogenic species occur
 Most of these are purely are environmental saprophytes

37. The medically important species
M. fortuitum
M. chelonae
M abscessus
M . smegmatis
M thermoresistible

38.  Pulmonary infections is uncommon
 Can cause local skin disease, osteomyelitis
 Can cause nosocomial disease
 Sources of M. fortuitum infection include implanted devices
such as catheters, injection site abscesses, and contaminated
endoscopes
 On LJ medium colonies can absorb the green dye
 No growth at 45°C, but grows on MacConkey agar

40.  Worldwide distribution.
 Found in tap water and other water sources.
 lung disease, joint infection, eye disease and other organ
infections.
 May result in non-healing wound, subcutaneous nodule or
abscess.
 Immunosuppression may cause disseminated lesions
throughout the body
 Increasing in patients with CF

41.  It was first isolated from gluteal abscesses in a 62-year-
old patient who had injured her knee as a child and had a
disseminated infection 48 years later
 Chronic lung disease, post-traumatic wound infections,
skin infections in immunodeficient patients, can be
associated with middle ear infections
 Produce arylsulfatase
 Shows tolerance to saline media (5% NaCl)
 Nitrate test negative
 Tween 80 negative

42.  First reported in 1981 as a human pathogen.
 Unique ability of the organism to grow at 52°C
 Pulmonary infection and cutaneous lesions
M .smegmatis
 Found in normal genital secretions (smegma)
 Generally considered a non-pathogenic
 Useful for the research analysis of other Mycobacteria species
in laboratory experiments

43. M .vaccae
 It is reported to be immunomodulation capable of inhibiting
tissue destroying hypersensitivity response and stimulating
protective immune processes in TB
 Clinical trials of M.vaccae vaccine in adjuvant to
chemotherapy in TB are underway
M .genevense ,M. confluentis
M. intermedium
 Non cultivable or poor growing NTM
 Characterised by 16S RNA base sequences

44. M. haemophilum
› Requires hemoglobin or hemin for growth in culture
› Will not grow on LJ or in automated system without the
addition of hemin supplements
› Painful subcutaneous nodules and ulcers, primarily in
AIDS patients or immune suppressed

45. We miss diagnosis of atypical mycobacterium on may occasions?
1.History and clinical examination
2.Radiographic features
3Bacteriolgic evaluation
a)Conventional diagnostic methods {smear, culture}
b)Immunologic diagnosis{ serological test ,T-cell assay e.g
tuberculin test ,INF gamma assay}
c)New diagnostic methods{ BACTEC, nuclic acid
amplification method…. }

46.  Sputum – 3 specimens - early morning
or at least 8 hours apart
 Bronchial lavage fluid
 Tissues or Wounds Swabs
 CSF or sterile body fluids
 Urine – 3 to 5 early morning collections
 Stool – M. avium complex only
 Gastric – for children
 Blood – disseminated disease

47. 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 cystic fibrosis sputum
specimens to eliminate the resistant (mucoid)
Pseudomonas strains
 Oxalic acid should not be used routinely, it is too harsh
and will decrease isolation of AFB

48. Carbol Fuchsin based stains
› Ziehl-Neelsen (ZN) – uses heat to drive stain into AFB
› Kinyoun – uses phenol to drive stain into AFB
› Read for 5 min on oil objective
Fluorochrome based stain
› Auramine Rhodamine – fluorescent stain, organisms stain
fluorescent gold, read on 25X or 40X for 2 min on a
fluorescence microscope
› Generally considered to more sensitive than ZN or Kinyoun

49. Solid media
Lowenstein-Jensen, Dorset Medium, Pawlowsky’s Medium
Ogawa, Tarshi’s Medium and Middlebrook 7H10/7H11
Liquid media
BACTEC TB-460
2- MGIT960 systems
Dubo’s media
Beck’s, Sula’s ,Sauton’s

50.  M. haemophilum, an iron source (ferric ammonium citrate or
hemin) has to be added to the medium, and the media are best
incubated at 30°C
 M. genavense, some success has been reported for media
composed of blood, charcoal, caseine, and yeast extracts,
acidified to pH 6.0
 M. marinum 30° C
 M. xenopi, M avium 42° C

51. 51
Growth on LJ Medium
Rapid growth within 7 days
Growth on MacConkey Agar
Aryl-sulphataes test
Slow Growth
Niacin test
-Ve
Tellurite Reduction
+ ve
M. smegmatis
- ve
M. phlei
- ve
Type of growth
+ ve
M.
tuberculosis
+ ve
M. Fortuitum
complex
Pigment Scanty Smooth
Flat Colonies
In Light
Group I
Photochromogens
In Dark
Group II
Scotochromogens
No Pigment
Group III
Non - Chromogens
- ve
BCG
+ ve
M. bovis
M.Kansasi
M.intermedium
M.Szulgai
M.scrofulaceum
M.Avium complex
M.gastri

52. 52
 QuantiFeron TB Gold Test.
 ELISA to detect 38 KDA
Mycobacterial Antigen.
 Molecular methods for early
diagnosis
* Amplicor test
* E – MTD
* DNA sequencing
* Line Probe Assay (LiPA)
* DNA micro arrays
* Molecular beacons.
* Single strand conformation
Polymorphism (SSCP)
* FRET Probes.
 Other PCR based
Techniques.
* Amplification Mutation
{ ARMS }
* Branch Migration Inhibition
{ BMI }
 Ligase Chain Reaction.
 Diagnosis of TB & Drug
Resistant TB with
Mycobacteriophages.

53.  Automated systems:
BACTEC MGIT 960 and BACTI-ALERT Instruments
› Use Liquid Middlebrook 7H9 tubed media for growth
› Both systems 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.
 BACTEC MGIT 960 NAP test
NAP = (p-nitro-α-acetylamino -B- hydroxypropiophenone)
TB does not grow in the NAP containing tube
NTM species grow in NAP

54.  The methods described most often are Pulse Field Gel
Electroforese (PFGE)
 Random Amplified Polymorphic DNA (RAPD).
 Amplified Fragment Length Polymorphism (AFLP)

55.  Gold standard for id of myco Molecular sequencing of genes
rpoB and hsp65.
 Nucleic acid probes .
 Nucleic acid amplification techniques.
 Bacilloscopy.
 Multiplex PCR.
 HPLC methods for NTM speciation

56. Principles of Treatment of NTM Disease
 The presence of the organism in a sterile site or
repeatedly from airway secretions in association with a
compatible clinical and radiologic picture confirms the
diagnosis.
 Treatment of rapidly growing mycobacteria should be
guided by in vitro susceptibilities. Other drug
susceptibility testing is not standardized.

57.  Treatment should usually combine at least two drugs of
proven efficacy.
 Contact follow-up is not necessary since NTM are not
transmitted from person to person.
 Duration of therapy has not been determined; in general, 6-12
months is required following negative cultures.

58.  Rifampicin, Ethambutol, Isoniazid, Minocycline,
Ciprofloxacin Clarithromycinn Azithromycin and
Cotrimoxazole.
 MAC=clarithromycin and ethambutol, sometimes
ciprofloxacin or rifabutin also (18-24mts)
 M. marinum species are often resistant to isoniazid. Treatment
with other antibiotics should be for at least two months
 M. kansasii treat for at least 18 months. clarithromycin
and rifampin

59.  M. chelonae .clarithromycin in combination with another
drug, Sometimes surgical excision needed
 Antibiotics are usually ineffective for M.ulcerans. Rifampicin
may promote healing of pre-ulcerative lesions. wound care
and removal of the necrotic tissue common.
 Surgical removal of infected lymph nodes and skin lesions
sometimes necessary.
 In severe cases, skin grafts may be necessary to repair the
surgical wound

60.  Satta et al.” treatment only based on expert opinion ,case
series and few clinical trial.”
 ELISA simple, fast ,high sensitivity and specificity. Current
large scaled multicentre study
 OPC67683 Anti-TB drug
 West Africa trial on M.ulcerans , combination of rifampin and
streptomycin daily for 8 wks
 Optimal drug combinations and dosing intervals for treatment
of pulmonary NTM.

61. 2012 Disseminated M .Marnium infection in hematopoietic stem
cell transplant recipient.

62.  M.Ulcerans-skeletal muscle contracture and atrophy.
 Male mice
 Injected near Right bicep muscle
 “The combination of BCG and HVJ-liposome/HSP65
DNA+IL-12 DNA by a prime-booster procedure could lead to
an overall effect of 100% survival in infected monkeys”.

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hairy cell leukemia. Am J Med 1986;80:891–896.
 Berger C, Pfyffer GE, Nadal D: Treatment of nontuberculous mycobacterial lymphadenitis with
clarithromycin plus rifabutin. J Pediatr 1996;128:383–386.
 Chaisson RE, Benson CA, Dube MP, et al: Clarithromycin therapy for bacteremic Mycobacterium
avium complex disease. A randomized, double-blind, dose-ranging study in patients with AIDS.
AIDS Clinical Trials Group Protocol 157 Study Team. Ann Intern Med 1994;121:905–911.
 Diagnosis and treatment of disease caused by nontuberculous mycobacteria. This official
statement of the American Thoracic Society was approved by the Board of Directors, March
1997. Medical Section of the American Lung Association. Am J Respir Crit Care Med 1997;156(2
Pt 2):S1–S25.
 Griffith DE, Girard WM, Wallace RJ Jr: Clinical features of pulmonary disease caused by rapidly
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64. THANK YOU