Mycobacterium

Dr.Gh.Hamzehloo

رخدادهایی که در سطح آزمایشگاه رخ می دهد و نتایجی که در پی

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تاخیر در تشخیص صحیح–مداخالت درمانی غیر الزم–عوارض نامطلوبی که در نتیجه ی در مان رخ داده بود–انواعی از دیگر آلودگیها، یافت شد– 200 ماه6ه ای ب6ود ک6ه در این بررس6ی 6حل این مش6کل نیازمن6د بررس6ی –

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تغییر در رویه های آزمایشگاه، مشکل را رفع نمود.–اش�تباهاتی ک�ه در آزمایش�گاهها رخ میدهن�د منج�ر ب�ه اتالف وقت، ان�رژی،

ن�ی�روی پر�س�نل میش�و�د و ن�ی�ز ب�ه ن�ت�ایج� آزم�ایش�ات اش�تب�اه را� ب�رای بیم�اران .ب��������ه ��������د�نب��������ال ��������خ�و�اه��������د� ��������دا�ش��������ت

Mycobacterium tuberculosis

Mycobacterium leprae (uncommon)

Mycobacterium avium-intracellulaire Complex (MAC) or (M. avium)

Important Human Pathogens

MYCOBACTERIUM• Aerobic bacilli –non spore forming

non motile

• Cell wall –rich in lipids

• Acid-fast bacilli

• Very slow growing

MYCOBACTERIA ASSOCIATED WITH HUMAN DISEASE

Mycobacterium Environmental contaminant Reservoir

M tuberculosis No Human

M bovis No Human, cattle

M leprae No Humn

M kansasii Rarely Water, cattle

M marinum Rarely Fish, water

M scrofulaceum Possibly Soil, water

M avium intracellulare

Possibly Soil, water, birds

M ulcerans No Unknown

M fortuitum Yes Soil, water, animals

M chelonae Yes Soil, water, animals

CLASSIFICATION OF MYCOBACTERIA ASSOCIATED WITH HUMAN DISEASE

Mycobacterium Clinical significance Pigmentation Growth

Unclassified

M Tuberculosis , M bovis

M ulcerans

Strict pathogens No No

M leprae Strict pathogen - -

Runyon Group 1

M marinum , kansasii

Runyon Group 2

Usually pathogenic Photochromogens slow

M scrofulaceum Rarely pathogenic Scotochromogens slow

Runyon Group 3

M avium intracellulare Pathogenic in immunocompromised

No slow

Runyon Group 4

M fortuitum, M chelonae Rarely pathogenic No ‘rapid’

Lipid-Rich Cell Wall of MycobacteriumMycolic acids

CMN Group: Unusual cell wall lipids (mycolic acids,etc.)

(Purified Protein Derivative)

Acid-Fast (Kinyoun) Stain of Mycobacterium

NOTE: cord growth (serpentine arrangement) of virulent strains

Photochromogenic Mycobacterium kansasii on Middlebrook Agar

NOTE: Mycobacteria pathogenic for humans can be differentiated (Runyon Groups) by:

speed of growth (all are slower than most other pathogens) and by

production of chromogenic pigments (in light, in dark, or none)

Improved Mycobacterial Isolation Medium

Pathogenic Mycobacterium spp.

BCG

AIDS patients

Mycobacterial Clinical Syndromes

Laboratory Diagnosis of Mycobacterial Disease

Nucleic acid probesNucleic acid sequencing

Differential Characteristics of Commonly Isolated Mycobacterium spp.

Mycobacterium tuberculosis

• Causes tuberculosis• Classic human disease• Pathogenesis• Transmission• Clinical presentations• Diagnosis• Treatment• Prevention

Mycobacterium tuberculosis

Mycobacterium tuberculosis

Infections

Mycobacterium tuberculosis

Infections (cont.)

BCG (bacille Calmette-Guerin) = attenuated M. bovis

Positive PPD + Chest X-Ray +

MDR-TB a serious global health threat

Pneumonia

Granuloma formation with fibrosis

Caseous necrosis• Tissue becomes dry & amorphous (resembling cheese)• Mixture of protein & fat (assimilated very slowly)

Calcification• Ca++ salts deposited

Cavity formation• Center liquefies & empties into bronchi

Typical Progression of Pulmonary Tuberculosis

Diagram of a

Granuloma

NOTE: ultimately a fibrin layer develops around granuloma (fibrosis), further “walling off” the lesion.

Typical progression in pulmonary TB involves caseation, calcification and cavity formation.

PPD Tuberculosis Skin Test Criteria

PPD = Purified Protein Derivative from M. tuberculosis

Chest X-Ray of Patient with Active Pulmonary Tuberculosis

Mycobacterium Tuberculosis Stained with Fluorescent Dye

Symptoms of Tuberculosis…

• may appear years after contracting the disease

• Fever

• Night-Time Sweating

• Loss of Weight

• Persistent Cough

• Constant Tiredness

• Loss of Appetite

Pathogenesis• Inhaled aerosols

Engulfed by alveolar macrophages Bacilli replicate Macrophages die

• Infected macrophages migrate local lymph nodes• Develop Ghon’s focus Primary complex• Cell mediated immune response

stops cycle of destruction and spread• Viable but non replicating bacilli present in macrophages

EVIDENCE OF INFECTION WITH M TUBERCULOSISChest x-ray / positive skin test

Case Finding

Most common tools for case finding include:

• History taking

• Physical examination

• Sputum examination

• X-ray examination

• Tuberculin skin testing

CLINICAL PRESENTATION

Pulmonary tuberculosisPulmonary tuberculosis

Primary complexAsymptomaticHEALS

REACTIVATIONPost-primary tuberculosis

Acute pulmonary diseaseSystemic spread

Asymptomatic /symptomatic

LATER DISEASERenal / CNS etc

MILIARY TUBERCULOSISPulmonarymeningitis

DIAGNOSIS

Pulmonary tuberculosisPulmonary tuberculosis

Primary complexAsymptomaticHEALS

REACTIVATIONPost-primary tuberculosis

Acute pulmonary diseaseSystemic spread

Aymptomatic /symptomatic

LATER DISEASERenal / CNS etc

MILIARY TUBERCULOSISPulmonarymeningitis

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1. Evidence of infection

a. Chest x-ray - hilar lymphadenopathy calcification of primary focus/LN

b. Delayed hypersensitivity response to purified protein derivative (PPD) MANTOUX /HEAF TEST

2. Evidence of active disease

a. Sputum for AFB positive

3. Evidence of active disease

a. Indirect evidence of infection (Mantoux)

b. Direct evidence of infection PCR / culture

c. Histo-pathological evidence

DIAGNOSIS

Acid-Fast Smear Showing TB Bacilli

© University of Alabama at Birmingham, Department of Pathology

Recording Sputum Smear Microscopy Results

Number of Acid-fast Bacilli

(AFB)

# of Oil Immersion Fields

Examine

Reported as:

No AFB Per 100 fields No AFB seen

(No AFB per 100 fields)

1-9 AFB Per 100 fields Scanty, record exact figure

(1-9 AFB per 100 fields)

10-99 AFB Per 100 fields 1+ (10-99 AFB per 100 fields)

1-10 AFB Per field 2+ (1-10 AFB per field in 50 fields)

More than 10 AFB Per field 3+ (>10 AFB per field in 20 fields)

Why the Emphasis on Sputum Smears?

Direct Microscopy is the most reliable and cost effective way to identify persons who are most likely to transmit TB

to others

© ITECH, 2006© University of Alabama at Birmingham, Department of Pathology

• Receipt of specimens: from clinics • Preparation and staining of smears• ZN microscopy /recording• Reporting of results• Maintenance of lab register• Management of reagents and supplies• Internal Quality Control (QC)• Collect specimen for culture and DST, send to

NTRL • Participation in EQA

Primary and District LabServices in TB control (Level 1)

Nyangagbwe Referral lab (Level 2)

• Activities: receive specimen for AFB and culture

• Services to clinics: FM/ZN smear microscopy (smear microscopy and send results)

• Support activities: (supply of reagents/ materials, training; EQA for smear microscopy including supervision)

• Inoculate specimen and refer to NTRL for incubation and DST

Role of NTRL in TB Control

• Identify mycobacterium other than MTB • DST of M. Tuberculosis• TB laboratory equipment services and

maintenance• Develop TB Lab manuals and guidelines• Primary link with NTP• Supervision of intermediate QA of culture and

microscopy• Operational and applied research• Provide EQA and monitor peripheral labs

Mycobacterial Culture (1)

• “Gold Standard” of TB diagnosis

• More expensive and more time consuming than microscopy

• Requires specialised training and media to perform

• Not recommended for routine case detection in Botswana

Courtesy of: Kubica G, 2007.

Eight Week Growth of Mycobacterium tuberculosis on

Lowenstein-Jensen Agar

Mycobacterial Culture (2)

Reasons to request mycobacterial culture:• Patient previously on anti-TB treatment• Still smear-positive after intensive phase of

treatment or after finishing treatment• Symptomatic and at high-risk of MDR-TB• To test fluids potentially infected with M.

tuberculosis• Investigation of patients who develop active PTB

during or after IPT• TB in health workers

• DST performed on all cultures– Tests for isoniazid, rifampicin, ethambutol,

and streptomycin

• If found to be multi-drug resistant, then send for additional testing for susceptibility to second-line medicines

TB Drug Susceptibility Testing (DST)

TREATMENT

• Anti-tuberculous drugs– INAH– Rifampicin– Ethambutol– Pyrazinamide

• DOT

• Multi-drug resistant tuberculosis

PREVENTION

• Incidence declined before availability of anti-tuberculous drugs

• Improved social conditions - housing /nutrition• Case detection & treatment• Contact tracing• Evidence of infection / disease• Treatment of infected / diseased contacts

ROLE OF IMMUNIZATIONBCG (bacillus Calmette Guerin)

ROLE OF IMMUNIZATIONBCG (bacillus Calmette Guerin)

حا 12 کیفی سیستم عاملهمیت ا ئز

کیفیت سیستماز ای مجموعه

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باشند می .کیفیت

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ی

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اسناد و مدارک

کنترل فرایند، کنترل

و کیفیتمدیریت

های نمونهآزمایشگاهی

و خریدی ذخیرهموجودی

ارزیابیمدیریت

رخداد

مدیریت

اطالعات

ی توسعهفرایند

خدمات مشتری

مدار

و تسهیالتایمنی

تجهی پرسنل سازمانزات

مدارو کاسناد

کاری جریان مسیر

04/18/23 44

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DO

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