# Presentation: 700592

## Mycobacterial Services

- Increasing transmission and antimicrobic resistance of  Mtbc
- & introduction of new technologies require adjustments in laboratory services

**Notes:**
This is the prompt slide for the Laboratory Diagnostic Tools presentation.

No Text Provided

## Safety in the Mycobacteria Lab

- Active TB in Mycobacteriologists
- 100 x General Population
-     3 x Other Laboratory Workers
- Skin Test Conversions in TB Labs
- 8-30%

**Notes:**
M. tuberculosis is a Class 3 pathogen
 i.e. an agent that may cause serious or potentially lethal disease as a result of expose by inhalation.  
Personnel handling such agents must have specific training in handling such organisms, be supervised by competent scientists, and work in physical facilities which minimize inhalation exposure.

## Safety in the Mycobacteria Lab (continued)

- Non-behavioral risk factors for infection
- Poor directional airflow
- Equipment failure
- Biological safety cabinets
- Centrifuges
- Autoclaves
- Specimens
- Number of positives processed
- Organism concentration

**Notes:**
Emphasize that the exposure to laboratory-generated aerosols while performing routine procedures is the most serious hazard encountered by lab personnel.  
Tell attendees that the laboratory must have special engineering and design features designed to contain and eliminate such aerosols.  
Similar to an isolation room there should be unidirectional air flow-- but in addition there needs to be biological safety hoods, and aerosol-containing centrifuges all of which must be frequently checked  to ensure proper operation.

Additional CDC/NIH References:
Biosafety in Microbiological &Biomedical Laboratories
Primary Containment for Biohazards:  selection, Installation and Use of Biological safety Cabinets.

## Safety in the Mycobacteria Lab (continued)

**Behavioral risk factors for infection**
- Lack of adequate safety guidelines
- Noncompliance with guidelines
- Aerosol production
-  Pouring, pipetting & mixing liquids
- Homogenizing  & cutting tissues
- Dropping or spilling culture flasks
- Preparing smears
- Needle sticks

**Notes:**
Significant aerosols are generated by:
	Pouring liquid cultures and supernatant fluids
	Fixed volume automatic puppeteers
	Mixing liquid cultures with a pipette
	Preparing specimens and culture smears
	Dropping tubes or flasks containing bacilli and 		homogenizing tissues 
	Breaking tubes during centrifugation.  

Since the onset of the HIV/AIDS epidemic, blood must now be considered a potential source of transmission of many pathogens.  

It is necessary for mycobacterium laboratories to develop adequate guidelines to minimize aerosol and blood exposures and properly instruct and supervise laboratory personal in their implementation. 

## Laboratory Biosafety Levels

**Biosafety Level - 2**
- Personnel trained in handling pathogens
- Lab directed by competent  scientist
- Access to lab is limited during work periods
- Extreme precautions with “sharps”
- Aerosol-producing procedures conducted in biologic safety cabinet

**Notes:**
During the 1970's a system was developed for categorizing etiologic agents into groups based on their mode of transmission, seriousness of illness resulting from infection, and availability of treatment and preventive measures.  Most common human pathogens such as S. aureus are considered class 2.  M. tuberculosis and C. immitis are class 3, and viruses such as Marburg and Ebola are class 4. 
Guidelines for appropriate facilities, containment equipment, procedures and work practices were developed to ensure organisms in each class would be handled safely.  These guidelines are referred to as Biosafety Levels (BL) 1 - 4. Mycobacterial specimen receipt, initial processing, the making and reading of mycobacterial smears, and the preparation of specimens for transport to a BL-3 facility for further work-up can be safely done in a biologic safety cabinet (BSC) if compliant with BL-2 conditions.  Other mycobacterial procedures, including concentration culture, identification procedures, susceptibility testing, and direct amplification of M. tuberculosis from clinical specimens should be handled under BL-3 conditions.

## Laboratory Biosafety Levels (continued)

**Biosafety Level - 3**
- Training in handling agents acquired by inhalation & able to cause  lethal disease
- Supervised by scientists experienced in working with such agents
- P3 laboratory facilities and procedures

**Notes:**
In a BL-3 (P-3) laboratory, more emphasis is placed on physical barriers to prevent contamination of the laboratory environment, and to protect personnel in contiguous areas and in the community from infectious aerosols. 
It is separated from other parts of the building by an anteroom with two sets of doors, or by access through a BL-2 area.
Air movement is unidirectional into the lab and all exhaust air from the area is directed outside the building without any recirculation.  
All procedures are carried out in a Class II or III BSC and personnel wear appropriate protective clothing and use equipment designed to contain or mitigate aerosol production.

## Levels of Laboratory ServiceATS Recommendations

- Level I
- Collect, smear & transport specimens
- Level II
- Smear & culture; ID & sensi testing of Mtb
- Level III
- Smear & culture; ID & sensi testing of all mycobacterial isolates

**Notes:**
In 1983, the American Thoracic Society (ATS) sanctioned the concept of “Levels of Service” in relation to the processing clinical mycobacteriology specimens. 
While the BLS recommendations, discussed above, are based on risks related to laboratory procedures, ATS’s “Levels of Service” relate to specimen load, personnel training and experience, quality of supervision, range of tests performed, availability of appropriate space and equipment, and regular, objective assessments of laboratory proficiency.  
Level I laboratories collect specimens, prepare & examine smears, and transport specimens to Level II or III laboratories.  
Level II laboratories perform AFB smears, set mycobacterial cultures, and identify &/or perform susceptibility tests on M. tuberculosis.   
Other mycobacterial isolates are forwarded to Level III laboratories for identification and susceptibility testing.

## Level I Laboratories

- Tasks
- Collect good specimens
- Prepare & examine smears
- Ship to Level II/III for culture & sensitivities
- Smear proficiency test scores of _>_ 80%

**Notes:**
Outlines the limited services that a Level I laboratory should provide.  
It should be emphasized that mycobacterial specimens sent to Level II/III laboratories must be packaged, labeled and shipped in accordance with Public Health Service (PHS), Department of Transportation (DOT), and International Air Traffic Association (IATA) regulations. 

## Level I Laboratories (continued)

- Characteristics
- Receive minimum of 10 AFB smears/week
- Use fluorochrome stain
- Quantitate smear AFB & report within 24 hr.
- Use certified biologic safety cabinet
- Send specimen for culture within 24 hr.
- Meet Dept. of HHS **B**iosafety **L**evel** 2    (BL-2) **requirements

**Notes:**
Describes the laboratory characteristics necessary for Level I service. 
 Importantly, they include BL-2 conditions, use a certified BSC,  read, quantitate and report smear results within 24 hours of specimen receipt, and send the specimen on for culture to a Level II/III laboratory within 24 hours. 
Ideally, they should use the more sensitive  fluorochrome stains. 

## Sputum Specimen Collection

- Collection of good specimen is critical
- First morning expectoration
- Rinse mouth with water
- Produce specimen by deep coughing
- Expectorate directly into lab-provided tube
- If specimen inadequate, induce sputum
- Dispatch immediately to lab
- Containers to be closed and sealed
- Refrigerate if not processed immediately

**Notes:**
This outlines the process of sputum specimen collection.  
Often clinical services, in their desire to rapidly determine the AFB smear status of a patient, will submit unsatisfactory specimens.  
They should be made aware of the necessity of obtaining a deep, expectorated specimen, ideally the first morning expectorate, to improve the likelihood of detecting respiratory tract mycobacteriosis.  
To minimize aerosol production in the laboratory, the specimen should be collected in a lab-provided, capped, 50 mm sterile plastic centrifuge tube, thereby eliminating the need to transfer the specimen for disinfection and concentration. 
When such a spontaneous expectorate can not be obtained, sputum induction or bronchoscopy should be utilized.

## AFB Smears

**Level I Laboratories**
- Recommended: specimen sterilization
- Open in BSC & sterilize with Clorox x 15 min
- Stain & read on open bench
- Specimen cannot be used for culture
- Probably safe - not currently recommended
- Open, prepare, dry & heat fix x 2h in BSC
- Stain & read on open bench
- Dispatch sealed container to Level II/III lab

**Notes:**
Describes the type of smear preparations that can be safely processed in Level I (BL-2) laboratory.  A 1995 CDC conference affirmed that if smears are made at BL-2, the specimen should be placed in a Class II or III BSC, opened, and a volume of 5% sodium hypochlorite (undiluted household bleach) equal to that of the specimen is added to sterilize the specimen.  After 15 minutes the specimen may be centrifuged and subsequently handled outside of the BSC.  The main disadvantage of this procedure is that the treated specimen can not be used for culture.

To avoid sterilizing the specimen, some authorities have recommended that, after placement in a the BSC, an bacteriologic loop or pipette can be used to remove a small aliquot of the specimen from its container, placed on a glass slide and heat-fixed at 65-75 C for 2 hours.  Although heat-fixed smears may still contain viable tubercle organisms, they are not readily aerosolized.  Personnel may, therefore, safely remove and stain the smears without taking additional precautions.  Moreover, the phenol contained in both traditional and fluorescent stains will kill any remaining viable organisms during the staining process.  It should be noted that, although direct smears are less sensitive than those prepared from concentrated specimens, they will detect most highly infectious sputum specimens.

## Level II Laboratories

- Tasks
- Perform all procedures done by Level I
- Isolate organisms in pure culture
- Identify M. tuberculosis complex
- Perform susceptibility testing of Mtb
- Refer MOTT isolates for ID & sensitivities
- Retain recovered isolates for 6 months
- Proficiency testing of isolation, ID and sensitivity procedures

**Notes:**
Outlines the services provided by Level II laboratories. 
These laboratories should enroll in proficiency testing programs that provide evaluation of their smear, culture, identification and sensitivity test procedures. 

## Level II Laboratories (continued)

- Characteristics
- Receive minimum of 20 specimens/week
- Use Class II or III biologic safety cabinet
- Detect growth in liquid media in 10 days
- ID with DNA probes, BACTEC NAP  or mycolic acid profile within 2-3 weeks
- Determine sensis of Mtb isolates in 3-4 w
- Meet Dept of HHS **B**iosafety **L**evel **3 (BL-3) **requirements

**Notes:**
Describes the laboratory characteristics necessary for Level II service.  
Importantly, they include BL-3 conditions and the use a certified Class II or III BSC. Smears and cultures are performed on concentrated specimens.  
If the lab receives less than 20 specimens weekly, it is unlikely that their personnel will be able to maintain their proficiency in isolation, identification, and susceptibility test procedures.  
To meet the listed “turn-around” criteria, they must utilize the newer technologies, including fluorescent microscopy, radiometric culture (BACTEC) systems or their equivalent, rapid identification methods (NAP test, DNA probes and/or HPLC for speciation, and radiometric susceptibility testing. 

## AFB Stains

**Concentrated Smear for Stain & Culture**
- Done in Level III laboratory BSC
- Liquify & decontaminate
- Centrifuge _>_ 3000G x 15min in sealed cups
- Prepare smear, dry, heat fix x2 h
- Stain and read within BL-3 facility
- Flourochrome (auramine) stains preferred

**Notes:**
This slide defines the procedure for concentrating respiratory specimens for smear and culture.

## Level III Laboratories

- Tasks
- Identify all mycobacterium
- Perform susceptibility tests on MOTTs
- Proficiency testing on MOTT identifications
- Work concentrated in  **very** **few** labs

**Notes:**
Level III laboratories differ from Level II in that they possess the capacity to identify and perform susceptibility tests on all mycobacterial isolates, not just those of M. tuberculosis.

## Level III Laboratories (continued)

- Characteristics
- Perform ~ 20 susceptibilities per month
- USE DNA probes, HPLC or other rapid ID methods
- Do not rely **only** on NA amplification for ID
- Meet Dept HHS **B**iosafety **L**evel** 3  (BL-3) **requirements

**Notes:**
A large specimen load is required to maintain proficiency in the procedures necessary for full mycobacterial services.  
Accordingly,  Level 3 services should be concentrated in relatively few laboratories.  
To ensure rapid turnaround times, Level 3 labs should utilize the newer technologies. 

## New Technologies

- Fluorescent microscopy
- Radiometric culture systems
- NAP test, DNA probes, HPLC
- Radiometric susceptibility testing
- Direct amplification of mycobacterial nucleic acid from specimens

**Notes:**
Fluorescent microscopy is approximately 15% more sensitive than the traditional Ziehl-Neelsen procedure.  Moreover, the contrast of the brightly-staining mycobacterial cells permits scanning the slide at magnifications as low as 250X, allowing the microscopist to examine the entire surface area of the smear in a relatively short period of time.  This is roughly 20X the surface area examined in 300 oif fields typically used with Ziehl-Neelsen stained smears.
Radiometric culture and susceptibility test procedures are considerably more rapid than traditional methods.  Moreover, radiometric cultures are more sensitive than traditional solid media (Lowenstein-Jensen and 7H-11) culture procedures.  Identification of mycobacterial isolates with DNA probes can be done in a few hours.  Although somewhat slower, the BACTEC NAP test and HPLC procedures are still considerably faster (and more reliable) than traditional biochemical identification methods. 

## New Technologies (continued)Direct Amplification Techniques

**AMTD approved for smear + respiratory specimens**
-  Requires 4-5 hours for completion
- Detects only M. tuberculosis complex
- Specificity approaches 100%
- Sensitivity 90-100%; less in smear negative specimens

**Notes:**
Direct amplification of mycobacterial nucleic acid in clinical specimens permits the specific identification of most patients with respiratory tuberculosis within a matter of a few hours. 

## AFB Stains

- Sensitivity on expectorated sputum
- M. tuberculosis		65%
- MOTT				20%
- Cum Sensitivity in series of 3 sputa
- M. tuberculosis		80%

**Notes:**
This slide along with the next 4 slides present data on smear and culture results from the Seattle VA Medical Center.  Although results from other laboratories may differ, the data provides a general picture of sensitivity and specificities of various mycobacterial procedures.
65% of all expectorated sputum specimens from which M. tuberculosis was isolated were smear positive; comparable data for specimens yielding Mycobacteria OTHER than Tuberculosis (MOTT) was only 20%.  
When a patient with culture-proven tuberculosis submitted 3 consecutive specimens for smear and culture, at least one of these specimens was smear positive 80% of patients.

## AFB Stains

- Cum sensi of smears in culture series that yield both a (+) culture for M. tuberculosis and one or more positive smears
- First specimen			  86%
- Second specimen			  95%
- Third specimen			  95%
- Fourth specimen			100%

**Notes:**
In patients who had a culture series in which yielded one or more positive cultures for M. tuberculosis and one or more positive smears, 86% of the tuberculous patients had a positive smear on the first specimen and 95% had a positive smear on the first or second specimen.  
Thus, 95% of tuberculosis patients who had one or more positive smears, and a positive smear on the first or second submitted specimen. 

## AFB Stains (continued)

**Specificity**
- Abs. False (+)		  0.05
- Rel. False (+)		  1.8
- Specificity			99.9
- PPV				98.1

**Notes:**
Only 1.8% of patients with a positive smear did not have a positive M. tuberculosis in the same series or an earlier pre-treatment series.  
The absolute false-positive rate was thus only 0.05%.  
The specificity of a positive smear was therefore 99.9 and the positive predictive value 98.1.  
These values are typical of smear results when utilizing techniques which diminish the possibility for cross-over contamination from one slide to another during the staining process. 

## Culture

**Notes:**
This slide demonstrates the culture positivity rate by specimen type.  
Thus 3% of all specimens yielded M. tuberculosis and 8% grew Mycobacteria Other Than Tuberculosis (MOTT).  
16% of blood specimens yielded a MOTT isolate; none grew M. tuberculosis. Interestingly, although the number of body fluids specimens were second only to those of respiratory specimens, the culture yield was extremely low.  
This raises the question of whether it is reasonable to continue to culture body fluid for mycobacterium unless there is strong supporting circumstantial evidence of body fluid infection.

## Culture (continued)

**Notes:**
This slide shows the sensitivity and time to positivity utilizing three culture media, the BACTEC radiometric liquid medium, and the 7H11 and Lowenstein-Jensen solid media.  
The BACTEC media was both the most sensitive (90%) and the speediest (an average of 12 days to positivity -- 20% were positive within 3 days).  
The BACTEC grew all M. tuberculosis isolates.  
In combination with either 7H11 or LJ, it had a sensitivity of 95-97%.  
The LJ  media was both the least sensitive (30%) and slowest (average of 34 days to positivity).

## CDC Isolation Guidelines

**Initiation of Isolation**
**Member of ‘high risk’ population**
-  Contact with infectious TB patient
-  Born in area of high TB prevalence
-  Medically under-served populations
-  HIV+, elderly, homeless, imprisoned
-  Alcoholics, IV drug users
-  **Appropriate clinical findings**

**Notes:**
These slides outline the CDC criteria for isolating a patient suspected of having infectious pulmonary tuberculosis.  
It should be noted that they require both evidence that the patient belongs to a high risk population and exhibits appropriate clinical or radiographic findings.  
This is important as patients are often isolated for tenuous reasons and the laboratory is asked to “rule out” infectious tuberculosis with a series of 3 negative smears and cultures.  
This is unnecessary if the rationale for isolation was obviously inappropriate and/or an alternate diagnosis compatible with the patients clinical status is criteria documented.

## CDC Isolation Guidelines (continued)

**Initiation of Isolation**
- Member of ‘high risk’ population
**Appropriate clinical findings**
-  Cough _>_ 3 weeks
-  Bloody sputum,
-  Anorexia, weight loss
-  Fever, night sweats

**Notes:**
No text provided

## CDC Isolation Guidelines

**Discontinuation of Isolation**
- Patients with proven infectious TB
-  Appropriate Rx (optimally 2-3w)
-  Clinical Improvement
-  3 consecutive negative smears (q2w)
-  Pt discharged to suitable locale
- Patients suspected of having infectious TB
-  Alternate diagnosis confirmed
-  Pt determined to be noninfectious

**Notes:**
If a patient has proven tuberculosis, the criteria for release from isolation are the initiation of therapy (optimally for 2-3 weeks), clinical improvement and 3 negative smears.  
Three consecutive negative smears are not required, however, if the patient satisfies the first two criteria, does not have a resistant organism, and is discharged to a suitable locale (home or a NH with isolation facilities).  
In fact, two thirds of our newly diagnosed tuberculosis patients are so discharged.

## Availability of AFB Smears

**CDC recommendations**:
- Results available within 24 hours
- No longer refer to “STAT” smears

**Notes:**
The CDC has dropped the term “STAT AFB” from its guidelines.  
It now suggests that laboratory services should provide smear results within 24 hours of receiving a suitable specimen.  
In Level I laboratories, this can usually be accomplished only with direct smears performed on sterilized specimens.

## The End

- Questions

**Notes:**
Check time available

## Timeline Comparison of TB MethodologyNew verses Old

- Specimen
- Received
-        Week           0                1                     2    3   4                     5      6     7     8     9   10   11   12   13   14   15   16   17    18
-                             ____________________________________________________________________________________
- Mycobacteria growth
- Bactec 460 System
- TB DNA Probe ID
- (after growth)
- Bactec Sensitivities
- (after growth)
- HPLC Atypical
- Mycobacteria ID
- Conventional Media
- growth
- TB Conventional Biochemical
- ID (after growth)
- Conventional Plate
- Sensitivitie
- Conventional Biochemicals

**     **
**    **** ****MTD**