Mycobacterium tuberculosis

Useful For

Diagnosis of respiratory and non-respiratory tuberculosis.

 

Testing Algorithm

Reporting Name Available separately Always performed
AFB microscopic NO YES
AFB culture NO YES
Mycobacterium multiplexed molecular assay NO YES
Drug susceptibility testing NO YES

 

*Mycobacterium multiplexed molecular assay performed on all non-fixed patient clinical specimens submitted to PHML for MTBC/Mycobacteria spp. testing. Refer to Mycobacterium multiplexed molecular assay for details

 

 

Method Name

Auramine fluorescent microscopy

 

Liquid and solid media culture (gold standard for diagnosis)

 

Drug susceptibility testing

 

Mycobacterium multiplexed molecular assay (Real-time polymerase chain reaction with fluorescent dye-labeled oligonucleotide probe detection).

 

 

Reporting Name

AFB Microscopy

Mycobacterium multiplexed molecular assay

TB Culture

 

Aliases

TB

Tuberculosis

AFB

Mycobacteria

General Specimen Requirements

  • All specimens should be collected in sterile, leak-proof, laboratory-approved containers.
  • Specimens should accompany a carefully completed requisition form providing the patient’s demographic data, the physician’s name, the date and time of collection, and the specimen type and site.
  • If possible, specimens collected for initial diagnosis should be obtained before the initiation of anti-TB therapy.
  • Once collected, specimens should be transported to the laboratory promptly.
  • Clinical specimens should be handled, processed and transported in an environment in which biosafety procedures are in place.
 

Specimen Type

 

  

Optimal Specimens

  

Transport/Storage

  

Rejection Criteria

  

Note
Sputum (spontaneous or induced) · At least three sputum specimens collected in 8 to 24 hour intervals, a minimum of one hour apart with at least one being an early morning specimen.

· Optimally 5-10mL each with 3 mL as a minimum

 · Transport to PHML as soon as possible at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light.

 · Saliva

· Pooled sputum

· Sputum < 3ml

 · If induced sputum specimen is collected, label the sterile container “INDUCED.”
Bronchoalveolar lavage or bronchial washing · Optimally 5-10 ml in sterile, leak- proof container

· Place the brush in a sterile, leakproof container with up to 5 ml of sterile saline

 · Transport to PHML as soon as possible at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light

 · Precious specimen

· Consult  Microbiologist on Call (MOC) if insufficient volume
Gastric lavage or wash

 

 

 

 

 

 

 · Collect specimens in early morning before patients eat and while they are still in bed

· Collect 5-10 ml in sterile, leak-proof container  containing 100 mg of sodium carbonate and mix well (contact Microbiology lab for container)

· One specimen per day on three consecutive days

 

 · Transport the specimen to the laboratory  as soon as possible at 2-8 °C

· Refrigerate the specimen if transport will be delayed.

 · Specimen that has not been neutralized within 1 hour of collection. · Consult MOC before collection to ensure appropriate specimen collection and timely processing
Abscess: closed or open, cellulitis, eye exudate, tissue, skin lesion · Specimen collection

– Closed abscess:
Remove surface exudate  by wiping with 70% alcohol. Collect fluid abscess material with a sterile syringe and remove tissue aseptically.

– Open lesion:
Aspirate material from under the lesion or abscess margin, if possible.

· Tissue (at least 1 g, if possible) in sterile, leak-proof container. Add 2-3ml of sterile normal saline to avoid dry up.

· Fluid abscess material in a sterile, leak-proof container.

 · Transport to PHML promptly at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light.

 · Abscess material submitted on a dry swab

· Swabs submitted in commercial swab transport devices or in transport gel–based medium

· Specimens submitted in formalin are unacceptable for smear and culture.

 · Swabs are generally not acceptable. Consult MOC before collection if it is the only specimen available. If approved, swabs should be submitted in 2–3 mL sterile saline.

· Abscess fluids collected by surgical procedure is a precious specimen.
Other body fluids (abdominal,
amniotic, ascites, bile, joint, paracentesis, pericardial,
peritoneal, pleural, synovial, thoracentesis)		 · As much as possible (recommended volume 10-15ml) in sterile, leak-proof container · Transport to PHML promptly at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light.

 · Specimen placed in formalin for histologic examination Precious specimen. Consult MOC if volume <2ml.

· Swabs are generally not acceptable. Consult MOC before collection if it is the only specimen available. If approved, swabs should be submitted in 2–3 mL sterile saline.
CSF · Optimally, collect 5-10 mL CSF (minimum volume 2 ml) in sterile, leak-proof container · Transport specimen as soon as possible at room temperature 

· Store specimens at room temperature. Do NOT refrigerate or freeze!

 · Precious specimen. Consult MOC if insufficient volume.
Tissue, lymph node biopsy · Collect as much as possible (at least 1g if possible) in sterile leak-proof container with 2-3 ml sterile saline to avoid dry up.  Do not wrap in gauze. · Transport to PHML promptly at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light.

 

 · Tissue or lymph node material submitted on a dry swab.

· Specimens submitted in formalin are unacceptable for smear and culture.

 · Precious specimen

· Consult MOC if insufficient volume.
Urine

 

 

 

 

 

 · Approximately 40ml urine. Minimum volume: 10mL

· First morning specimen preferred

· Collect 1 specimen per day on 3 consecutive days (clean catch, midstream method)

 · Transport to PHML promptly at 2-8 °C.

· Storage: If transport is delayed, refrigerate specimen and protect from light.

 · Pooled urine

· Urine from catheter bag

· Urine in preservatives

 · Urine should be tested only if renal or urinary tract Mycobacterial infection is suspected.
Blood

 

 

 

 

 

 

 

 

 

 

 · Collect two Myco/F lytic blood culture bottles 30 min apart

· Optimal volume: 3-5 mL; the range of blood volume: 1-5ml

 · Transport specimen as soon as possible at room temperature 

· Store specimens at room temperature. Do NOT refrigerate or freeze!

 · Blood collected in EDTA, which greatly inhibits mycobacterial growth even in trace amount.

· Coagulated blood

· Serum or plasma

 · Consult MOC for Myco/F lytic blood culture bottles shipped to ward.

· Blood culture is Indicated usually in immunocompromised patients with systemic infection.
Bone Marrow Collect bone marrow in a sterile, leak-proof container. · Transport specimens as soon as possible at room temperature.

· Store specimens at room temperature. Do not refrigerate or freeze bone marrow specimens.

 

 · Precious specimen
Consult Microbiologist on Call for other specimen types not included in the table.

 

Tuberculosis (TB) is one of the top 10 causes of death worldwide and a leading killer of HIV-positive persons. In 2018, Mycobacterium tuberculosis caused approximately 1.5 million deaths and accounted for 10 million newly diagnosed cases globally. In Canada, in 2017 there were 1,796 cases of active tuberculosis reported. Two populations — foreign-born individuals and Indigenous Peoples — accounted for the majority of cases. 1,290 cases of active tuberculosis disease were reported among foreign-born individuals and 313 cases reported among Canadian-born Indigenous Peoples. Canadian-born, non-Indigenous people accounted for 125 cases.

 

M. tuberculosisis spread from person-to-person via respiratory transmission, and has the potential to become resistant if proper antimycobacterial treatment is not administered. Rapid and accurate detection of M tuberculosisin patient specimens is of clinical and public health importance.

 

Interpretation

AFB Microscopic

 

The threshold of detection of AFB in concentrated specimens using flourochrome stain is 5,000 – 10,000 bacteria/ml of sputum. The specificity of the AFB smear is high for mycobacteria (ALL nontuberculous mycobacteria will be AFB positive). Rarely organisms, such as Nocardia and Actinomycetes, can be weakly acid-fast. A positive AFB smear almost always indicates the presence of mycobacteria, but not necessarily M. tuberculosis.

 

Number of bacteria seen on microscopy and laboratory interpretation:

 

Flourochrome (250X magnification) Laboratory Report
0 in 30 fields Negative
1-2 per 30 fields Report exact number
1-9 per 10 field 1+
1-9 per field 2+
10 – 90 per field 3+

 

TB PCR

 

Refer to Mycobacterium multiplexed molecular assay for details

 

 

AFB culture

 

As few as 10 – 100 viable bacteria can be detected by culture.

 

Solid and liquid cultures are incubated for 8 weeks and 6 weeks respectively before being reported as NEGATIVE.

Procedure Days Turnaround time Specimen Retention
AFB Microscopic Mon – Sun 24 hr 8 weeks
TB PCR Mon – Sun 48 hr 8 weeks

 
Culture Mon – Sun 8 weeks 8 weeks

 

 

Methods Description

 

AFB Microscopic: Auramine fluorescent microscopy

Mycobacterium multiplexed molecular assay

Culture:

1) BACTTEC MGIT Liquid Media

2) LJ Solid Media

 

 

Performing Laboratory Location

 

Public Health Microbiology Laboratory

100 Forest Road

St. John’s

NL A1A 3Z9

 

 

References

 

  1. Forbes et al. Practice Guidelines for Clinical Microbiology Laboratories: Mycobacteria, Clinical Microbiology Reviews, 2018.
  1. Canadian Tuberculosis Standards 7th Edition: 2014.