Confirming the Diagnosis of Tuberculosis using Microbiology.

Hi every body.

During the exams both the FCPS 2 and the MBBS many students do not have any idea as to how to confirm the diagnosis of TB and have very wrong ideas. The gold standard is to demonstrate Mycobacterium tuberculosis or a bacterium resembling it in the test material.

What is the test material that we usually test? Sputum (not saliva so be careful about specimen collection), gastric aspirate preferably on an empty stomach so fasting is a good option and it gives better results in very young children who swallow their own sputum, discharge from a cutaneous sinus, pleural aspirate, peritoneal aspirate, biopsy material before it is put into formaldehyde, pericardial fluid, CSF and fluid from a joint aspiration. Please note that I am not listing blood as an option as TB is a localised disease, bacteremia is transient when it occurs in miliary TB and is over by the time we make a diagnosis so trying to obtain AFB (acid fast bacteria) from blood is not an option we ever exercise in clinical practice. I am not discussing TB osteomyelitis here.

Staining for mycobacteria and why they are called “acid fast”. Mycobacteria are Gram positive i.e they stain purple or blue with Giemsa stain. This staining method is not used as they cannot be distinctly identified from conventional bacteria. The mycolic acid structure of the cell wall of mycobacteria confers the ability to resist destaining by acid or alcohol after being stained by certain aniline dyes, leading to the term acid-fast bacillus (AFB). Microscopy to detect AFB (using Ziehl-Neelsen or Kinyoun stain) is the most commonly used procedure to diagnose TB in the world, especially in developing countries with limited laboratory capacity. Sadly third world physicians specially in Pakistan do not use this simple method as extensively as should be done. Please remember to do so in your own practice. Learn the details of these two staining methods.

A specimen must contain at least 104 colony forming units (CFU)/mL to yield a positive smear, hence a negative result does not mean that AFB are not present. Please repeat the test at least 3 times at intervals. A fluorochrome dye (such as auramine O) provides an easier, more efficient, and approximately a 10-fold more sensitive alternative. Sputum smears will be positive if 10,000 AFB are present per ml i.e a sputum specimen with 9,999 mycobacteria may be negative!!! As microscopic detection of mycobacteria does not distinguish M. tuberculosis from nontuberculous mycobacteria, hence the lab results are written “AFB resembling mycobacteria seen”.

Culturing Mycobacterium tuberculosis. Remember they grow slowly,  (a conventional or phenotype culture will take 3-8 weeks with a resistance report will come in another 3-8 weeks) and do not grow on culture media used for conventional bacteria.

There are three types of traditional culture media: egg based (Lowenstein-Jensen), agar based (Middlebrook 7H10 or 7H11), and liquid (Middlebrook 7H12 and other commercially available broths). Growth in liquid media is faster (generally one to three weeks). Growth tends to be slightly better on egg-based medium, but growth is more rapid on agar medium. Agar medium permits examination of colony morphology and detection of mixed cultures.

Two manual liquid medium systems, the Mycobacteria Growth Indicator Tube (MGIT) and MB Redox tube systems are available and a the radiometric BACTEC-460 semiautomated system for recovery of Mycobacterium tuberculosis from sputum specimens. The Bactec gives a growth index of 15 in 2 weeks. The MODS (microscopic observation of drug susceptibility) allows the detection of resistance to rifampicin and isoniazid ie MDR or multi drug resistance TB. This is also a liquid medium.

Molecular tests — Molecular methods are available for detection of M. tuberculosis complex DNA and common mutations that are associated with drug resistance. There are two major types of molecular assays: probe-based (non-sequencing) tests and sequence-based assays. All results from molecular methods need to be confirmed with rapid culture methods because the molecular methods do not distinguish between dead or viable bacteria.

NAAT. Nucleic acid amplification test. This is a form of PCR. it can detect 1-10 organisms per ml of sputum and can distinguish between tuberculous and non-tuberculous mycobacteria. A positive NAA result supports the diagnosis of TB in the appropriate clinical and epidemiologic circumstances; smear positivity together with positive NAA is considered sufficient for diagnosis of tuberculosis. A negative NAA result is not sufficient to exclude the presence of active TB or drug resistance. Two assays are available. The Amplified MTD test is approved for smear-positive or smear-negative respiratory specimens from patients with suspected pulmonary TB and fewer than seven days of treatment; it detects TB but does not detect drug resistance. The Xpert MTB/RIF assay is approved for only induced or expectorated sputum from untreated patients or patients on fewer than 3 days’ therapy; it detects mycobacteriumTB and rifampin resistance.

Urine LAM antigen detection in HIV infection — Urine-based detection of mycobacterial cell wall glycolipid lipoarabinomannan (urine LAM) assay is an assay for diagnosis of TB in patients with HIV.

What is an “open case of TB”? This term usually refers to a patient with active pulmonary TB, who is coughing up sputum which contains AFB which may have been detected by staining methods or repeated slides still need to be seen. Patients who have pulmonary TB and are coughing up sputum are potential open cases. If AFB have been demonstrated in their sputum they are highly infected. They should be asked to wear a mask which is frequently changed, and kept in a ward where immune suppressed patients are not exposed to them and young children are kept away from them. If facilities are available they should be kept in a separate ward with reverse barrier nursing for at least 4 weeks preferably 6 week. Why this period? If effective anti=TB therapy has been started the AFB though still present in the sputum are no longer viable or able to colonise hence the chance of infecting other people are very low. With Rifampicin this period is reduced to 2 weeks. Be careful with the multi drug resistant forms of AFB.

Rapid Growing Mycobacteria. Mycobacteria are very slow growing except for certain varieties called the rapid growers. RGM  are also called non-tuberculous mycobacteria.

  • M. fortuitum causes human infection primarily by direct inoculation, causing abcesses, surgical wound infections, and catheter-related sepsis . Rarely, it can cause keratitis, prosthetic valve endocarditis, cervical lymphadenitis, and pulmonary disease.  Look for it in patients with bronchiectasis.
  • M. chelonae,  infection in immunosuppressed patients, including hematogenously disseminated disease. M. chelonae may also cause surgical wound infections and keratitis .
  • M. abscessus is the most pathogenic of the RGM group and the RGM most likely to cause pulmonary infection, primarily in patients with underlying lung disease such as cystic fibrosis related and non-cystic fibrosis related bronchiectasis. Two main sub-species are found: bolletii (resistant to macrolides) and maselliense.

AFB in stool. The stools contain many saprophytic mycobacteria hence the detection of mycobacteria in a stool smear is not diagnostic of intestinal TB.

Published by


I am a Professor of Medicine and a Nephrologist. Having served in the Army Medical College, Pakistan Army for 27 years I eventually became the Dean and Principal of the Bahria University Medical and Dental College Karachi from where I retired in 2016. My passion is teaching and mentoring young doctors. I am associated with the College of Physicians and Surgeons Pakistan as a Fellow and an examiner. I find that many young doctors make mistakes because they do not understand how they should answer questions; basically they do not understand why a question is being asked. My aim is to help them process the information they acquire as part of their education to answer questions, pass examinations and to best take care of patients without supervision of a consultant. Read my blog, interact and ask questions so that I can help you more.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s