Acid Amplification Test
Dr.M.P. Samrat Abhishek
District TB Control
Born Robert Heinrich
11 December 1843
Kingdom of Hanover
Died 27 May
1910 (aged 66)
Grand Duchy of Baden
•Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis and, less
commonly, by other organisms of the ‘tuberculosis complex’. It is estimated that 3 million
people die from TB each year—the majority of them in developing countries.
• The annual incidence of New cases of all forms of TB (pulmonary and extra-pulmonary)
worldwide is estimated to be approximately 8 million, of which about 95% occur in
developing countries. Many TB cases in developing countries remain
undiscovered. Of the discovered smear-positive cases, less than half complete
• Consequently, the estimated prevalence (the total number of tuberculosis cases
at a given time) worldwide is 16 to 20 million, of whom about 8 to 10 million are
sputum smear-positive and highly infectious.
•The number of persons infected with the tuberculosis bacillus is estimated to be 1.7
billion, of which 1.3 billion live in developing countries. In India, more than 40% of adults
are infected with TB, and approximately 1.5 million cases are put on treatment every
year. An estimated 5 lakh deaths from TB occur every year.
INCIDENCE OF TB IN INDIA
o Tuberculosis is one of the deadliest public health
threats today, but there remains a lack of effective
o untreated tb patients remain a source of infection for
other members of community & results in
considerable morbidity & mortality , especially in
Screening methods for tuberculosis:
The most widely used method to detect TB is
sputum smear microscopy test, which has a
number of drawbacks including……
• low sensitivity (especially in HIV-positive individuals
• inability to determine drug-susceptibility
• variable performance that depends on operator
X-Ray especially the digital version serves as the
most sensitive tool but only shows opacities
which is further depends upon the x-ray reader(inter
& intra reader variations of reporting).
Conventional diagnosis of drug resistant TB
relies on mycobacterial culture and drug
susceptibility testing (DST), a slow and
cumbersome process requiring sequential procedures
for isolation of mycobacteria from clinical specimens.
Identification of Mycobacterium tuberculosis
complex, and in vitro testing of strain susceptibility to
During this time patients may be inappropriately
treated, drug-resistant strains may continue to
spread, and amplification of resistance may occur.
The CBNAAT/ Xpert MTB/Rif test is a cartridge-
based fully automated NAAT (nucleic acid
amplification test) for TB case detection and
Rifampicin resistance testing.
Purifies, concentrates, amplifies (by rapid, real-time
PCR) and identifies targeted nucleic acid It sequences
in the TB genome (rpo b), and provides results from
unprocessed sputum samples in less than 2 hours,
with minimal hands-on technical time.
Diagnosis of TB, TBHIV, MDR-
TB and XDR-TB WITH CBNAAT :
Sputum smear microscopy has a particularly low
sensitivity for detecting TB among PLHIV.
This is because people in later stages of HIV infection and
with compromised immune systems often release fewer
organisms into their sputum, at concentrations
below the threshold for visual detection under a
For PLHIV with a negative smear microscopy result
but who are still presumed to have TB, bacterial
culture has been the other option.
culture can only be undertaken at central level
laboratories, and results are normally only available
after a number of weeks or months.
Culture is therefore not good enough for people living
with HIV, who need a speedy TB diagnosis and
CBNAAT allows for the rapid detection of TB and
resistance to rifampicin in a single test.
It has a sensitivity superior to that of conventional
microscopy or culture on solid media, and is therefore
useful in the diagnosis of TB in HIV co-infected
persons where the sensitivity of microscopy
alone is low.
o The system simultaneously detects resistance to
rifampicin, which is a good and reliable proxy for
who guide line:
WHO recommends the use of Xpert MTB/RIF as a
primary diagnostic test for all people living with
HIV who have signs and symptoms of TB.
For people with unknown HIV status presenting with
strong clinical evidence of HIV infection.
For people who are seriously ill and suspected of
having TB regardless of HIV status and those at high
risk of MDR-TB.
WHO has issued policy recommendations for the use of Xpert MTB/RIF in the
diagnosis of extrapulmonary TB and rifampicin resistance detection
• Xpert MTB/RIF should be used in preference to conventional microscopy and
culture as the initial diagnostic test in testing cerebrospinal fluid specimens
from patients presumed to have TB meningitis (strong recommendation given
the urgency of rapid diagnosis, very low quality of evidence);
• Xpert MTB/RIF may be used as a replacement test for usual practice
(including conventional microscopy, culture, and/or histopathology) for testing
of specific non-respiratory specimens (lymph nodes and other tissues) from
patients presumed to have extrapulmonary TB (conditional recommendation,
very low quality of evidence).
•For CSF specimens, Xpert MTB/RIF should be preferentially used over culture if the
sample volume is low or additional specimens cannot be obtained, in order to reach
quick diagnosis. If sufficient volume of material is available, concentration methods
should be used to increase yield;
•Individuals presumed to have extrapulmonary TB but with a single Xpert MTB/RIF -
negative result should undergo further diagnostic testing and hence processing of
tissue samples (lymph nodes and other tissues) for Xpert MTB/RIF should include a
decontamination step to enable samples to be concurrently cultures
•Pleural fluid is a suboptimal sample for the bacterial confirmation of pleural TB, using
any method. A pleural biopsy is the preferred sample.
•These recommendations do not apply to stool, urine or blood, given the lack of
data on the utility of Xpert MTB/RIF on these specimens.
CBNAAT SUSPECTS CRITERIA:
A. All PLHIV cases.
B. All MDR close contacts .
C. All retreatment smear negative cases & extra
D. All Peadiatric TB suspect cases.
E. Referral cases from public private partnership.
Samples that are recommended for CBNAAT
Sputum- BAL , Gastric lavage (with out food particles).
FNAC / BIOPSY material.
Mid stream urine.
Stool & blood are not recommended.
Lymph nodes / biopsy: Tissue sample pieces grinded
in a sterile container (tissue grinder).
CSF sample of 0.1 ml to 1 ml is recommended for
cbnaat. Less than 0.1 ml is insufficient sample.
Pleural fluid & asitic fluid of 5 ml is recommended for
Blood stained & xanthochromic samples may cause
All specimens should be transferred to the DTC as
early as possible preferred in a stored condition at 2 –
8 degree centigrade.
The Xpert MTB/RIF assay can be used directly for CSF
specimens and homogenised extrapulmonary samples (lymph
node biopsies and other tissues) or on decontaminated
specimens if culture is performed concurrently.
Whenever possible, specimens should be transported and stored
at 2 to 8°C prior to
processing (a maximum of 7 days).
LPAs are currently limited to detecting resistance to
rifampicin and isoniazid and are suitable only on
smear positive Specimens.
Conventional DST is needed to detect resistance to
anti-TB agents other than rifampicin and isoniazid &
to detect XDR-TB.
Overall the LPA test is a reliable, rapid and easy to
perform for the simultaneous detection of RMP and
INH resistance in M.tuberculosis
It is recommend that the LPA should serve as an early
guidance of therapy, which should be followed by a
phenotypic DST confirmation for all suspected MDR-
Although Xpert MTB/RIF is suitable for use at all
levels of the health system & does not require
additional laboratory equipment, it requires care in
handling, a stable and uninterrupted electrical supply,
security against theft, adequate storage space & bio-
Xpert MTB/RIF does not eliminate the need for
capacity for conventional TB microscopy, culture and
The Xpert MTB/RIF assay is suitable for diagnosing
TB and detecting RR-TB.
A negative result accurately excludes the possibility of
rifampicin resistance and no further testing is needed
to confirm the negative results. (NPV>99%)
Microscopy or culture, or both, remain essential for
monitoring treatment since molecular tests based on
DNA detection have not been shown to be suitable.
Liquid culture, molecular LPAs and the Xpert
MTB/RIF assay should be phased in to programmes in
a way that ensures that the existing capacity for solid
culture and DST is maintained.