Tuberculosis (TB), Multi Drug Resistant TB (MDR TB) and Extreme Drug resistant TB (XDR TB) ! Diagnosis, Culture and Susceptibility Testing An Update
Dr C Heney - September 2006

Saliva is not an appropriate specimen! The patient should rinse his mouth with water to decrease bacterial contamination. For patients with an unproductive cough, an induced specimen with the help of a physiotherapist may be required. Gastric aspirates are useful in children who are unable to produce sputum.

Patients should submit 2 to 3 early morning sputa on different days. The first specimen has an 83% yield. The second specimen increases the yield by 10 - 12%.

Direct microscopy is useful -
· In the diagnosis of TB
· During treatment to monitor progress
· At the end of treatment to establish cure.

Two specimens should be sent for microscopy prior to the end of the intensive phase of treatment and 2 specimens after the completion of 5 months of treatment.

Note that patients with advanced HIV disease have an increased incidence of smear-negative TB.

Results are quantified as negative, 1+ (scanty), 2+ (moderate) or 3+ (innumerable).

Smear negative TB patients
In immuno-suppressed patients who often have paucibacillary disease, the smears may be negative as the organisms are present in such low numbers. Culture still remains the gold standard for the diagnosis of TB.

Culture
Culture for TB increases the sensitivity of diagnosis by 20 - 40%. Culture is performed using the MGIT technique (Mycobacterial Growth Indicator Tube). The MGIT has an oxygen sensitive fluorescent sensor  embedded at the base and actively growing and respiring mycobacteria consume the dissolved oxygen, unmasking the fluorescence which is detected by the sensor.
The increased yield in the MGIT is due to the bigger innoculum size used and the addition of the nutritional supplement.

Time to positivity
Smear-positive specimens can be detected within 4 - 14 days on average.
Smear-negative specimens take longer, usually about 3 weeks.
All cultures are incubated for a maximum of 6 weeks to improve detection.

Confirmation of MGIT positive cultures A ZN stain is done to confirm this 'positive' culture. Species identification is performed using molecular techniques. The Gen-probe uses innovative genetic probe technology to identify the genetic finger print of Mycobacterium tuberculosis by detecting rRNA directly and rapidly. This test is specific for the mycobacterium tuberculosis complex. This includes M. tuberculosis, M. bovis, M. bovis-BCG and M. africanum. M. bovis infections are rare except in areas with high transmission of M. bovis from animals to
humans. M. bovis-BCG may be detected after adverse reactions to the BCG vaccination. M. africanum rarely causes human disease. The Genotype PCR identifies the M. tuberculosis
complex.

Susceptibility testing
With the current increase of TB in South Africa and the problems of MDR and XDR TB, it is probably advisable to perform first-line susceptibility testing on all culture-positive specimens. Routinely, TB microscopy and culture are performed only. Because of the expense, TB susceptiblity testing is done by request only unless specifically indicated, as "TB CULTURE
AND SENSITIVITY" on the request form. This excludes cash patients on whom either 'microscopy only' or 'microscopy and culture only' are done as indicated on the request form.

· The following drugs can be tested. These are Rifampicin, INH, Ethambutol, Pyrazinamide and
Streptomycin

· Rifampicin resistance rarely occurs in isolation and is usually found in combination with INH
resistance.

· At present the test is performed using the MGIT. Depending on the rate of growth of the
organism, a result is obtained within 2 weeks. In the near future, susceptibility testing will be
performed using PCR technology which detects specific genes encoding for resistance. This will improve the turn-around time and decrease the costs of the tests.

· Rifampicin and INH resistance implies 'Multi Drug Resistant TB' or MDR-TB. Only then is
second-line testing indicated.

· Another exciting innovation is the FASTPlaque-Response test. This is a rapid bacteriophage assay for the determination of rifampicin resistance in sputum specimens containing the mycobacterium tuberculosis complex. It can be performed directly on the sputum and results are available within 48 hours on a smear-positive specimen which is a t least 1+ positive. This screening test is being is being introduced shortly and all positive results will be confirmed by
conventional susceptibility testing. MDR-TB probably accounts for at least 1% of all new
cases and 5% of re-treatment cases. MDR TB is difficult and expensive to treat with a cure rate of <50%.

MDR TB should be suspected if:
· The sputum still has the same amount of positivity on microscopy at the end of the 2 months of intensive treatment
· The sputum is still positive at the end of the continuation phase
· Clinical treatment failure and interrupted cases
· Close contacts of MDR TB patients

Second-line drugs tested include kanamycin, ofloxacin and ethionamide. Currently, these tests are performed at the NHLS using the MGIT system but with the increase in resistance, Lancet plans to introduce these tests in the near future.  MDR-TB resistant to 3 of the second-line drugs is by definition 'Extreme Drug Resistant TB' or XDR-TB.

The Tuberculin test has limitations because of the high burden of TB in our patient population. It must be interpreted taking into account the clinical picture, BCG status, the age of the patient (it is unreliable in those over 5 years of age), HIV status and known contact with an infectious TB patient. A positive reaction occurs after BCG immunization. A positive result indicates infection with TB but not necessarily TB disease. A negative skin test does not exclude TB.

New screening tests
If the patient has been previously exposed to M. tuberculosis, their T-cells will secrete the cytokine interferon gamma into the plasma. This can be measured by the Quantiferon Elisa Test. It is a simple whole blood assay with results available within 24 - 48 hours. The Quantiferon test requires special tubes which can be ordered from the lab. This test will not be able to differentiate active disease from latent infection but will differentiate TB infection from BCG and TB infection from nearly all non-TB mycobacterial infections.

The current commercially available TB Antibody detection tests are screening tests only. They should be used with caution and in conjunction with other diagnostic and clinical information until further international and local recommendations are made available.

For more information, please contact:

LANCET LABORATORIES Pretoria:
Switchboard: (012) 483 0100
Client services: (012) 483 0110

LANCET LABORATORIES Johannesburg:
Switchboard: (011) 358 0800
Client services: (011) 358 0888

LANCET LABORATORIES Kwa-Zulu Natal:
Switchboard: (031) 308 6500
Client services: (031) 308 6655