MLAB 2534  CLINICAL MICROBIOLOGY

MYCOBACTERIA

  CHARACTERISTICS

• SLOW GROWING BACILLI; NON-MOTILE
• AEROBIC
• NON-SPORE FORMING
• ACID FAST
• HIGH LEVELS OF LIPID IN CELL WALL (MYCOLIC ACID)
• ISOLATION MAY TAKE 8 WEEKS
• GRAM STAINS POORLY BECAUSE THE LIPIDS INTERFERE WITH THE CRYSTAL VIOLET AND SAFRANIN
• ACID-FASTNESS IMPORTANT PROPERTY THAT IS DEPENDENT ON ITS CELL WALL; ORGANISMS THAT DO NOT DECOLORIZE ARE DESCRIBED AS “ACID FAST”…..MYCOBACTERIA ARE ACID FAST BACILLI (AFB)
   

MAJOR GROUPINGS

• MYCOBACTERIUM TUBERCULOSIS COMPLEX WHICH  OCCURS IN HUMANS AND CAUSES  TUBERCULOSIS INFECTIONS
• AND NONTUBERCULOUS MYCOBACTERIA (NTM) – NUMEROUS SPECIES; ARE ALSO REFERRED TO AS MOTT (MYCOBACTERIA OTHER THAN TUBERCULOSIS OR ATYPICAL MYCOBACTERIA
   

CLINICAL SIGNIFICANCE

• HISTORICALLY DEVASTATING DISEASE
• KNOWN AS CONSUMPTION AND HAS BEEN KNOWN IN ALL AGES AND CLIMATES
• PROGRESS HAS BEEN MADE TO CONTROL
• 1.7 BILLION PEOPLE INFECTED IN THE WORLD
• MORE THAN 20,000 CASES REPORTED ANNUALLY IN THE UNITED STATES
• RECENT INCREASE IN RESISTANT STRAINS
• IMMUNOCOMPROMISED HOSTS, AIDS AND IV DRUG ABUSERS
• OTHER FACTORS- NONCOMPLIANCE TO TREATMENT, COMPLICATIONS AND SOCIAL FACTORS (MULTI-DWELLING, HOMELESSNESS)

CLINICAL SIGNIFICANCE AND IDENTIFICATION OF ACID FAST BACILLI

MYCOBACTERIUM TUBERCULOSIS (MTB)

• PRIMARY AGENT OF HUMAN TUBERCULOSIS (TB) AND MOST IMPORTANT SPECIES
• RESPIRATORY TRACT IS MOST COMMON SITE OF PRIMARY TB
• PRIMARY TB IS AN INFECTION IN THE PREVIOUSLY UNINFECTED PERSON
• TRANSMITTED THROUGH THE INHALATION OF AIRBORNE DROPLETS (COUGHING)
• GROWS AND MULTIPLIES IN LUNGS
• VIABLE ORGANISM STAYS IN BODY FOR DECADES
• WHEN HOST BECOMES DEBILATED, THE LATENT ORGANISMS RE-EMERGE CAUSING SECONDARY TUBERCULOSIS

• NOT PART OF RUNYON GROUPS
• SLOW GROWER
• ROUGH PALE COLORED COLONIES
• NIACIN POSITIVE
• NITRATE POSITIVE
• CATALASE NEGATIVE
• 97% PROBABILITY

MYCOBACTERIUM BOVIS

• OCCASIONALLY CAUSES TB-LIKE SYMPTOMS IN HUMANS
• TYPICALLY INFECTS CATTLE AND OTHER ANIMALS

• VERY SLOW GROWING - GROWS IN 21 DAYS

• GROWTH NONPIGMENTED

• NIACIN NEGATIVE

• NITRATE NEGATIVE
   

MYCOBACTERIUM AFRICANUM
 

• FOUND IN TROPICAL AFRICA AND CAUSES TUBERCULOSIS
   

NONTUBERCULOUS MYCOBACTERIA

            CLASSIFICATION OF MYCOBACTERIA OTHER THAN TUBERCLE BACILLI ON BASIS
             OF  GROWTH RATE, PIGMENTATION AND PHOTOSENSITIVITY OF PIGMENTATION
 

• GROUP I - PHOTOCHROMOGENS
• DEVELOP YELLOW PIGMENT WHEN EXPOSED TO LIGHT SOURCE -
• NONPIGMENTED IN DARK
• TAKE MORE THAN 7 DAYS TO GROW
• TABLE 48-3
                         
• GROUP II - SCOTOCHROMOGENS
• PIGMENTED, YELLOW TO ORANGE, WHEN GROWN IN DARK OR LIGHT
• TAKE MORE THAN 7 DAYS TO GROW
• TABLE 48-4
   
• GROUP III - NONPHOTOCHROMOGENS
• NONPIGMENTED REGARDLESS OF LIGHT OR DARK
• TABLE 48-5
   
• GROUP IV - RAPID GROWERS
• GROWS IN 3 TO 5 DAYS
• SELDOM PATHOGENIC
• TABLE 48-6
   

NONCULTIVATABLE NTM - MYCOBACTERIUM LEPRAE

• CAUSE OF LEPROSY (HANSENS DISEASE)
• DISEASE OF SKIN, MUCOUS MEMBRANES AND NERVE TISSUE
• CANNOT BE CULTIVATED ON ARTIFICIAL MEDIA
• PCR USED TO IDENTIFY IN INFECTED TISSUES
• ARMADILLOS ARE NATURAL RESERVOIR AND FOOTPADS OF MICE

LABORATORY SAFETY

• SAFETY IS AIMED AT PREVENTING AEROSOLS
• BIOLOGICAL SAFETY HOODS
• PROPER VENTILATION
• PERSONNEL SAFETY
• PROPER DISINFECTANT
• OTHER PRECAUTIONS
   

SPECIMEN COLLECTION

• MYCOBACTERIA CAN BE RECOVERED FROM A VARIETY OF SPECIMENS
• SUCCESSFUL ISOLATION BEGINS WITH PROPERLY COLLECTED AND HANDLED SPECIMENS
• SPUTUM AND OTHER RESPIRATORY SECRETIONS
• GASTRIC ASPIRATES AND WASHINGS
• URINE
• STOOL
• BLOOD
• TISSUE AND OTHER BODY FLUIDS
• WOUNDS, SKIN LESIONS AND ASPIRATES
   

INAPPROPRIATE SPECIMENS

• SWABS
• 24 HOUR POOLED SPUTUMS
• 24 HOUR POOLED URINES OR STOOLS

SPECIMEN PROCESSING

• CONCENTRATION
• DECONTAMINATION
• DIGESTION

REAGENTS USED

• N-ACETYL-L-CYSTEINE + 2% NAOH  (PROCEDURE PAGES 564 -565)
• SODIUM HYDROXIDE
• BENZALKONIUM CHLORIDE
• OXALIC ACID

ACID-FAST STAINS AND PROCEDURES (PAGE 566)

• ZIEHL-NEILSON
• CARBOLFUCHSIN WITH HEAT
• ACID ALCOHOL DECOLORATION  (95% ETHANOL AND 3% HCL)
• COUNTER STAIN WITH METHYLENE BLUE
• EXAMINE UNDER OIL (1000X)
  
   
• KINYOUN CARBOLFUCHSIN
• DOES NOT REQUIRE HEAT
• 3.0% ACID-ALCOHOL DECOLORIZER -(70% ETHANOL AND 0.5% HCL)
• METHYLENE BLUE
• EXAMINE UNDER OIL

• FLUOROCHROME
• USES FLUORESCENT DYES
• AUROMINE O
• RHODAMINE B
• STAINS EVEN NON-VIABLE CELLS
• LIPID IN CELL WALL MAKES AFB RESISTANT TO DECOLORIZATION EVEN WITH ACID
• EXAMINE UNDER LOW POWER FOR FLUORESCENCE

EXAMINATION AND INTERPRETATION OF SMEAR

·         REFER TO PAGE 678  BOX 22-3

CULTURE MEDIA AND ISOLATION METHODS

• AGAR-BASED MEDIA
• MIDDLEBROOK 7H10 AND 7H10 SELECTIVE
• MIDDLEBROOK 7H11 AND 7H11 SELECTIVE
  
   
• EGG-BASED
• LOWENSTEIN-JENSEN IS THE MOST COMMON
• L-J  WITH GRUFT, PYRUVIC ACID OR IRON
  
   
• LIQUID
• BACTEC 12B MEDIUM
• MIDDLEBROOK 7H9 BROTH
• SEPTI-CHEK AFB
• INCUBATE FOR 10 DAYS
• SHORTER INCUBATION PERIOD
  
   
• CULTURES INCUBATED AT 35°C IN DARK
• ATMOSPHERE 5% - 10% CO2
• ANOTHER SET AT 25°C
• CULTURES EXAMINED WEEKLY FOR GROWTH
• AFTER 8 WEEKS REPORTED AS NO GROWTH
• CONTAMINATED CULTURES SHOULD BE DISCARDED

OTHER IDENTIFICATION TESTS

• NIACIN
• NITRATE REDUCTION
• CATALASE
• TWEEN 80 HYDROLYSIS
• TELLURITE REDUCTION
• ARYLSULFATASE
• THIOPHENE-2-CARBOXYLIC ACID HYDRAZIDE (TCH) INHIBITION
• OTHER TESTS SUCH AS NUCLEIC ACIDS

THERAPY

• MOST COMMON DRUG COMBINATION IS INH AND RIFAMPIN FOR 9 MONTHS
• IF PYRAZINAMIDE IS ADDED LENGTH IS SHORTENED TO 6 MONTHS
• FINALLY INH PROPHYLAXIS

PREVENTION

• BCG VACCINE – ONLY AVAILABLE AT PRESENT
• PRESENTLY FOUR OTHER TYPES OF VACCINES ARE BEING EVALUATED IN ANIMALS