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Lab Guide – 2019 TB Section Lab Guide
2
CLINICAL
MYCOBACTERIOLOGY (NATIONAL TB
REFERENCE LABORATORY)
SPECIMEN COLLECTION
GUIDE
3
NATIONAL TB REFERENCE LAB – HGH SPECIMEN GUIDELINES
Critical value (CRIV) in TB Reference Lab
Mycobacteriology (TB) Test
Critical Result (Record the time of Identification)
Acid Fast Bacilli smear Smear positive result from direct specimen in TB Lab
First GeneXpert PCR test
First GeneXpert positive result, where smear results are negative
First TB Culture First culture positive result from direct specimens in TB lab, where GeneXpert and smear results are negative
For Collection guideline
CLINICAL MYCOBACTERIOLOGY SPECIMEN COLLECTION GUIDE
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1. Abscesses and aspirates
ITEM PROCESS
Cerner order AFB smear/AFB PCR/ C AFB.
Specimen requirement
Decontaminate the surface site with 70% alcohol.
Collect purulent material aseptically from an undrained
abscess using a sterile needle and syringe. Open abscess
(after incision and drainage) and collect the expressed
material with a needle and syringe.
Transfer 5-10 ml of aspirated fluid abscess material or Pus
aspirate in 50 ml sterile conical tube (leak proof without
fixative).
Pus aspirate is always superior to a swab specimen. Specify
anatomic Site.
Volume 5-10 ml. (Minimum 2 ml)
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative
cultures 42 days; for positive culture, when culture
becomes positive the preliminary report is released within
24 hours of detection time. Final result for MTB sensitivity is
differing from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Average number of AFB / 100 fields, >100/field.
Culture: Growth of MTB complex with susceptibility to
primary drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
Rejection criteria Swab specimen (only smear done).
Swabs are a not suitable because they dry easily and because of the limited amount of material transferred.
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Swabs are not optimal for AFB culture and PCR.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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2. Body Fluids
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Disinfect site with 70% alcohol if collecting by needle and syringe. Aspirate fluid as much as possible optimal volume (10 ml desirable) in a 50 ml sterile conical tube leak proof container without fixative.
Specify anatomic site (Plural, Pericardial, Peritoneal, Abdominal, Amniotic, Ascites, Bile, Synovial, and OTHERS).
Volume Minimum 10-15 ml.
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures 42 days; for positive culture, when culture becomes positive the preliminary report is released within 24 hours of detection time. Final result for MTB sensitivity is differing from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100 fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
Rejection criteria
Swab dipped in fluid.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
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Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
3. Bone marrow
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
MGIT BD tube obtained from TB lab.(Discards half of the
media and add the sample).
Or 10 ml yellow top collectors containing polyanethol
sulfonate (SPS).
Transport Temperature ≤ 2 hrs. as soon as possible at Room Temperature.
Do not refrigerate or freeze Bone marrow specimen.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative
cultures 42 days; for positive culture, when culture
becomes positive the preliminary report is released
within 24 hours of detection time. Final result for MTB
sensitivity is differing from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
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Rejection criteria
Bone marrow specimen sent in unsterile tube.
Bone marrow specimen in EDTA tube, plain tube or any other
tube than SPS tube.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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4. Cerebrospinal fluid (CSF)
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement Collect aseptically 2-3ml CSF Sterile leak-proof sterile tubes.
Volume 2 to 3 ml, optimal is upto10 ml.
Transport temperature ≤ 2 hrs, as soon as possible at room temperature.
Do not refrigerate or freeze CSF specimen.
Days test performed Daily.
Turnaround time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures 42 days; for positive culture, when culture becomes positive the preliminary report is released within 24 hours of detection time. Final result for MTB sensitivity is differing from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary
drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
Rejection criteria Specimen sent in unsterile tube.
Specimen in tube with preservative or additive.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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5. Gastric wash or lavage
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Collect three consecutive days in early morning before
patients eat and while are still in bed.
Perform lavage with 25 to 50 mL of chilled, sterile distilled
water. Recover sample and place in 50 mL conical sterile
tube.
Volume 25 to 50 mL (Recommended 15ml).
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary
drugs SIRE AND PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
Rejection criteria Gastric lavage sent on swab.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
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Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
6. Sputum specimens
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Rinse mouth with water to remove food particles, debris,
mouthwash, or oral drugs, which may inhibit the growth of
Mycobacteria.
Collect sputum (for 2 consecutive days), have patient
breathe deeply and cough several times to achieve a deep
lower reparatory specimen. (not postnasal fluid) .
volume Collect 5-10 ml (minimum 3ml early morning specimen).
Transport temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42. PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to
primary. Drugs SIRE AND PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
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Rejection criteria
Salivary sputum except for patient on therapy in the TB unit
and induced sputum.
Third consecutive sputum from the same patient, pooled
sputum.
Leaking specimens and swabs.
Specimen for culture, submitted in un-sterile containers.
Swabs are not optimal for AFB culture and PCR.
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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7. Lower Respiratory specimens
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Lower Respiratory Endo-tracheal, Trans-tracheal secretions,
Bronchoalveolar Lavage or Bronchial wash in a 50 ml sterile
conical tube leak proof container without fixative.
Induced sputum is collected by trained staff.
For induced sputum, use sterile hypertonic saline. Avoid
sputum contamination with nebulizer reservoir water.
Saprophytic Mycobacteria in tap water may produce false-
positive culture or smear results. Indicate on test order if
specimen is induced sputum, as these watery specimens
resemble saliva and risk rejection as inadequate.
Avoid contaminating bronchoscope with tap water when
collecting Bronchoalveolar Lavage or Bronchial washing.
volume Collect 5-10 ml (minimum 3ml early morning specimen).
Transport temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42. PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to
primary. Drugs SIRE AND PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
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Rejection criteria
Third consecutive pulmonary samples from the same patient.
Leaking specimens and swabs.
Swabs are not optimal for AFB culture and PCR.
Specimen for culture, submitted in un-sterile containers.
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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8. Stool
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Collect specimen in a clean bed pan or use plastic wrap
placed between the toilet seat and the bowl. Do not submit
feces contaminated with urine or toilet water.
Stool specimen in clean leak proof wide mouth container
without preservative. (Preferably 50 ml conical tube).
Utility of culturing stool for acid-fast bacilli remains
controversial and should be discouraged; however, stool
samples from immunocompromised and HIV patients may be
submitted, mainly to detect MOTT.
The stool specimen from patient other than mentioned above
requires prior consultation with TB Lab.
Volume 10-20 gm (minimum 1g).
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No grow at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no of AFB / 100 fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
16
Rejection criteria
Stool in transport medium or preservatives.
Stool samples from NON immunocompromised and HIV patient.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
17
9. Tissue / biopsy
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Tissue collection is an invasive procedure and requires a
trained physician. Include material from both the center and
the edge of the lesion.
Aseptically collected tissues in 50 ml sterile containers
without fixatives or preservatives.
Specify anatomic site.
Volume > 1 ml or >1g (submit as much as possible).
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report of Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary
drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected
Rejection criteria
Tissue specimen sent in fixatives like formalin.
Swab specimen. Swabs are not optimal for AFB culture and
PCR.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
18
10. Urine for AFB
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
First morning specimen in sterile container (midstream is
never advised). Organisms accumulate in bladder overnight,
so first void morning specimen provides best yield.
Specimens collected at other times are dilute and are not
optimal.
Collect urine specimen on 3 consecutive days.
Volume 15-20 ml (prefer up to 40 ml).
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary
drugs SIRE and PZA or growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
19
Rejection criteria
24 h pooled specimen AND small volume.
Urine from bag.
Urine received in Boric acid container.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
20
11. Wound specimen
ITEM PROCESS
Cerner order AFB smear/ AFB PCR/ C AFB.
Specimen requirement
Collect Tissue or pus aseptically in 50 ml sterile conical tube in leak proof without fixative and avoid indigenous microbiota.
Select caseous portion if available as the majority of organisms will be found in the periphery of a caseous lesion. Do not wrap in gauze. Freezing decreases yield.
Specify anatomic site.
Volume > 1 ml or > 1 gm.
Transport Temperature As soon as possible. Refrigerate the specimen at 2-8 °C.
Days test performed Daily.
Turnaround Time
Microscopy and AFB PCR: 24 hr. and/or Next day
Exception: outpatient samples and extra pulmonary
specimens received on Thursday and weekends, will be
released on Sunday.
Culture: No specific Turnaround time, for negative cultures
42 days; for positive culture, when culture becomes positive
the preliminary report is released within 24 hours of
detection time. Final result for MTB sensitivity is differing
from case to case.
Method Microscopy: Auramine O and Ziehl-Neelsen.
Culture: BACTEC MGIT.
GeneXpert TB PCR.
Reference value Microscopy: Negative, No AFB seen.
Culture: No growth at 42 days.
PCR: Negative, MTB DNA not detected.
Interpretation
Microscopy: Report Average no. of AFB / 100
fields,>100/field.
Culture: Growth of MTB complex with susceptibility to primary
drugs SIRE and PZA or Growth of MOTT.
PCR:
Positive, MTB DNA Detected Rif resistance Detected.
Positive, MTB DNA Detected Rif resistance Not Detected.
21
Rejection criteria
Specimens in fixatives or preservatives e.g. tissue specimen
submitted in formalin.
Specimen for culture, submitted in un-sterile containers.
Leaking containers
One swab for multiple requests, for example, for various
organisms (bacteria, AFB Fungi, Virus etc.)
Performing Lab Location National TB Reference Laboratory – Rumailah, Ext. # 6671 / 6675.
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