Chapter 22 Mycobacterium tuberculosis Other Nontuberculous

Chapter 22 – Mycobacterium tuberculosis & Other Nontuberculous Mycobacteria MLAB 2434 – Clinical Microbiology Cecile Sanders & Keri Brophy-Martinez

General Characteristics ¢ ¢ Slender, slightly curved or straight rod-shaped organisms Non-motile Do not form spores Cell wall with extremely high lipid content Staining requires longer time or application of heat l Once stained, resist decolorization with acid-alcohol (acid-fast) l

General Characteristics (cont’d) ¢ ¢ ¢ Strictly aerobic Grow more slowly than most bacteria Traditional characteristics used to identify Mycobacterium l l l Rate of growth Colony morphology Pigment production Nutritional requirements Optimum incubation temperature Biochemical test results

General Characteristics (cont’d) ¢ Newer techniques Automated culture system, such as BACTEC l Nucleic acid probes with PCR l Thin-layer chromatography l GLC l High-performance liquid chromatography l

Safety Considerations ¢ Mycobacteriology workers are three times more likely to seroconvert (develop positive skin test) l l l Adequate safety equipment Safe laboratory procedures training Information on hazards Preparations for unexpected accidents Staff must be monitored regularly by medical personnel

Safety Considerations (cont’d) ¢ Skin test Also called “Mantoux test” and PPD l Those with positive skin test must be advised to have chest X-ray l ¢ Proper Ventilation Separate from other parts of lab l Negative air pressure (6 to 12 room air changes/hour) l

Safety Considerations (cont’d) Biological Safety Cabinet ¢ Use of Proper Disinfectant ¢ Bactericidal for mycobacteria l Also called “tuberculocidal” l ¢ Other precautions Disposables l Protective clothing, face masks l

Specimen Collection and Processing Variety of clinical specimens, including respiratory, urine, feces, blood, CSF, tissues, and aspirations ¢ Should be collected before antibiotic therapy and processed ASAP ¢ Sputum is most common; should be collected in a wide-mouth container to avoid aerosols ¢

Specimen Collection and Processing (cont’d) ¢ Sputum Number of specimens needed is inversely related to the frequency of smear positivity l Should be from a deep cough or expectorated sputum induced by neubulization l Bronchial washings or lavages may be collected l

Specimen Collection and Processing (cont’d) ¢ Gastric aspirates Used to recover mycobacterium that may have been swallowed during the night l Only used when patient is unable to produce a good quality sputum specimen l ¢ Urine – First morning preferred

Specimen Collection and Processing (cont’d) ¢ ¢ ¢ Stools – primarily collected from AIDS patients to determine Mycobacterium avium complex (MAC) Blood – most commonly from AIDS and other immunosuppressed patients Tissues and other body fluids – need a fairly large volume of CSF, since number of organisms in that site are rare

Digestion & Decontamination of Specimens ¢ ¢ Because Mycobacterium grow so slowly and are often collected from non-sterile body sites, they are easily overgrown by other bacteria Specimens from non-sterile sites, therefore, must be “decontaminated” Sputums or other viscous specimens also must be “digested” Specimens from sterile sites (CSF, etc. ) do not need decontamination

Digestion & Decontamination of Specimens (cont’d) ¢ Decontamination l l Specimen from non-sterile site is mixed with an agent that will kill nonmycobacterium bacteria Common decontamination agents • Na. OH is most common • Benzalkonium chloride (Zephiran) • Oxalic acid l After decontamination, the agent must be neutralized so that it will not eventually kill the Mycobacterium

Digestion & Decontamination of Specimens ¢ Digestion Liquefying mucus enables the mycobacterium to contact and use the nutrients in the agar medium l Common digestion agents l • N-acetyl-L-cysteine – most common • Trisodium phosphate (Z-TSP) – used with Zephiran

Concentration ¢ After decontamination and digestion, the specimen is centrifuged in a closed, vented centrifuge to concentrate the organisms

Acid Fast Stains ¢ ¢ ¢ After centrifugation, the button at the bottom of the tube is used to make a smear and to inoculate media Acid Fast Stains l Ziehl-Neelsen – uses heat to drive the color into the lipids of the cell wall; decolorized with acidalcohol l Kinyoun – cold stain l Auramine or auramine-rhodamine fluorochrome stain – more sensitive After staining, a minimum of 300 oif are examined

Culture Media and Isolation Methods Mycobacterium are strictly aerobic ¢ Slow growers; cultures held for 6 weeks before calling negative ¢ Media ¢ Lowenstein-Jensen (LJ) media – egg based l Middlebrook 7 H 10 and 7 H 11 agar – serum based l Middlebrook 7 H 9 broth l

Culture Media and Isolation Methods (cont’d) ¢ Labs with large volumes of Mycobacterium cultures use an automated reader (BACTEC) BACTEC broth contains 14 C-labeled substrate l When organisms grow, 14 C in the form of 14 CO 2 is released and detected radiometrically l

Laboratory Levels or Extents of Service for Mycobacterium ¢ American Thoracic Society levels 1. Specimen collection only l 2. Acid-fast stains and/or inoculation only l 3. Isolation and presumptive identification of Mycobacterium species l 4. Definitive identification and antibiotic sensitivity testing l

Identification of Mycobacterium ¢ Colony Morphology l ¢ Either smooth and soft or rough and friable Growth rate Rapid growers – colonies in < 7 days l Slow growers – colonies in > 7 days l ¢ Temperature

Identification of Mycobacterium (cont’d) ¢ Photoreactivity Photochromogens – produce carotene pigment upon exposure to light l Scotochromogens – produce pigment in light or dark l Nonchromogenic – no pigment; these colonies are a buff color l

Identification of Mycobacterium (cont’d) ¢ Biochemical Identification l l Most labs now use nucleic acid probes with or without PCR Older tests • • • Niacin accumulation Nitrate reduction Catalase Hydrolysis of Tween 80 Iron uptake Arylsulfatase

Identification of Mycobacterium (cont’d) l Older tests (cont’d) • Pyrainamidase • Urease • Inhibitory tests • • • NAP TCH Growth in 6. 5% Na. Cl Tellurite reduction Growth on Mac. Conkey

Antibiotic Sensitivity Testing for Mycobacterium ¢ ¢ ¢ These tests must be performed with great attention to detail, because Mycobacterium is fairly resistant and only a few organisms left can cause reinfection Development of drug-resistance Common antibiotics (usually two or more are given) l Isoniazid l Rifampin l Ethambutol l Streptomycin l Pyrazinamide

Mycobacterium Infections ¢ Truly pathogenic l l l ¢ Potential pathogens l l ¢ M. tuberculosis M. bovis M. ulcerans M. kansasii M. marinum Other possible pathogens and rare pathogens listed on p. 670

Mycobacterium tuberculosis Primarily a pathogen of the respiratory tract (“TB”) ¢ One of the oldest communicable diseases ¢ Over 1 billion cases worldwide, with 8 to 10 new cases each year and 3 million deaths per year ¢ Once called “consumption” ¢

Mycobacterium tuberculosis (cont’d) ¢ Primary tuberculosis l l Spread by coughing, sneezing, or talking Inhaled into alveoli, where the organisms are phagocytized If the organism does not cause immediate infection, the organism can be “walled off” in a granuloma Granulomas can break down in future and the organisms can cause infection later

Mycobacterium tuberculosis (cont’d) ¢ PPD Test

Mycobacterium tuberculosis (cont’d) ¢ PPD Test (cont’d) Positive Test

Mycobacterium tuberculosis (cont’d) ¢ Extrapulmonary tuberculosis Spleen l Liver l Lungs l Bone marrow l Kidney l Adrenal gland l Eyes l

Mycobacterium tuberculosis (cont’d) ¢ Identification Slow grower l Colonies are thin, flat, spreading and friable with a rough appearance l May exhibit characteristic “cord” formation l Grows best at 35 to 37° C l Colonies are NOT photoreactive l

Mycobacterium tuberculosis (cont’d)

Other Mycobacteria ¢ ¢ Mycobacterium bovis – primarily in cattle, dogs, cats, swine, parrots and human; disease in humans closely resembles M. tuberculosis MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous mycobacteria) l l Most found in soil and water Chronic pulmonary disease resembling TB

Other Mycobacteria (cont’d) ¢ NTM (cont’d) M. avium Complex (MAC) l M. kansasii l Mycobacterium fortiutum-chelonei Complex l M. marinum l Etc. , etc. l

Mycobacterium leprae ¢ ¢ ¢ Causes leprosy or Hansen’s Disease Infection of the skin, mucous membranes and peripheral nerves Most cases are from warm climates Bacteria infect the cooler areas of the body (ears, nose, eyebrows, fingers, toes) Diagnosis made from finding acid-fast bacilli in scrapings from lesions Not culturable, except in mouse foot pads

Mycobacterium leprae (cont’d)