Indian Pediatr 2014;51: 1007-1011
Is Xpert MTB/RIF Assay in Gastric Lavage
Aspirate Useful for Diagnosis of Smear-negative Childhood
Source Citation: Pang Y, Wang Y, Zhao S, Liu J,
Zhao Y, Li H. Evaluation of the Xpert MTB/RIF assay in gastric lavage
aspirates for diagnosis of smear-negative childhood pulmonary
tuberculosis. Pediatr Infect Dis J. 2014;33:1047-51.
Section Editor: Abhijeet Saha
This study evaluated the Xpert MTB/RIF assay for
diagnosis of smear-negative childhood pulmonary tuberculosis (TB) using
gastric lavage aspirates (GLA) in 211 Chinese children. The sensitivity
in detecting children with a clinical diagnosis of TB for MGIT and Xpert
was 12.1% (95% CI 9.3%, 14.9%) and 48.6% (95% CI 44.4%, 52.8%),
respectively. The authors concluded that Xpert MTB/RIF assay is an
excellent tool for the diagnosis of smear-negative childhood with GLA
samples. The high proportion of very low mycobacterial load in the GLA
samples from smear-negative TB cases may increase the frequency for
obtaining indeterminate RIF resistance results by Xpert.
Relevance: Childhood tuberculosis poses
unique diagnostic challenges different from adults. These are related to
paucibacillary infection, absence of hallmark symptoms/signs such as
hemoptysis or a pulmonary cavity on radiography, overlap of clinical
symptoms with various differential diagnoses, logistic/technical
challenges in obtaining sufficient quantity of appropriate biological
specimens and poor specificity of Mantoux test. These challenges create
debates on whether to ‘prove and treat’ or ‘treat and prove’ the
condition. Although many physicians give a therapeutic trial when in
doubt, the practice has also led to indiscriminate prescription of
anti-tuberculosis drugs (ATD) resulting in inadequate/insufficient
anti-tuberculosis therapy (ATT), thereby contributing to the problem of
drug resistance. Therefore, it would be welcome to have diagnostic tests
that are sensitive enough to identify tuberculosis disease (as opposed
to infection) and specific enough to lead to correct therapeutic
decisions (starting or stopping therapy). The GeneXpert MTB/RIF assay
(Cepheid, Sunnyvale, CA), has been suggested as a test fulfilling these
criteria . The test received a shot in the arm when the World Health
Organization (WHO) recently recommended [2,3] that it may (note
emphasis) be used as the initial test in children with suspected
tuberculosis instead of attempting to identify/detect Mycobacteria
through smear and culture. WHO also recommended it as the first test for
children with suspected multi-drug resistant tuberculosis or disease
associated with HIV infection, however data on these conditions in
Indian children are lacking. A recent Cochrane review  also suggested
that the test is very promising both as an initial diagnostic test as
well as an add-on test in those who are smear negative. However, the
review did not examine studies in children. The Revised National
Tuberculosis Control Programme (RNTCP) has chosen a more cautious
position and merely lists it as one of the tests endorsed by it . In
2012, the Indian Academy of Pediatrics expert committee rightly
emphasized that all efforts should be made to detect Mycobacteria
through smear, culture, or the Xpert assay . Against this backdrop,
the recent study by Pang, et al.  evaluating the diagnostic
utility of the test in gastric lavage samples of children with suspected
TB but negative smear examination, is highly relevant.
Critical appraisal: Table I
summarizes the methodological aspects of the study . Additional
points to be considered are as follows. Ideally, Xpert MTB/RIF assay
should be shown to be comparable with the reference standard (either
culture or a clinical diagnosis leading to treatment). However, this
study shows that it is inferior to culture and perhaps clinical
diagnosis (Table I), although the latter is relatively
non-specific. Inferiority to culture has been demonstrated in several
other studies as well [9-11]. This is surprising because the principle
of nucleic acid amplification tests (NAAT) is to detect even miniscule
amounts of nucleic acids (through amplification), hence theoretically
NAAT are expected to be more sensitive than culture. This is the basis
for the high sensitivity of polymerase chain reaction (PCR) based
diagnosis of infection (and superiority over culture). In this study
, the Xpert MTB/RIF assay failed to identify 6/17 (35%) smear
negative, culture positive cases. Some or all of these 17 children may
not have fulfilled the criteria for ‘clinically diagnosed TB case’ as
per the authors’ definition. Therefore, they would not have received
treatment until the culture results became available. This suggests that
reliance on Xpert MTB/RIF instead of smear and culture as the initial
diagnostic test could result in non-treatment of these children. Rather
than exploring the relatively poor sensitivity of Xpert MTB/RIF assay,
Pang, et al. have chosen to downplay it citing comparable results
in other studies. This poses a real danger that in future also, this
serious limitation of the assay will simply be ignored.
Table I Critical Appraisal of the Study Validity
Are the results of the study valid?
The investigators applied the index test (Xpert MTB/RIF assay)
in 211 children with suspected tuberculosis and compared the
test results against two reference standards. Children who
were smear positive (n=15) were not included. Tuberculosis was
suspected in the presence of any one of the following: cough
longer than 2 weeks, fever lasting beyond 2 weeks, weight loss
(magnitude undefined), history of contact with TB (undefined)
and suggestive radiography. It is unclear whether eligible
participants were enrolled consecutively or an element of
selection bias existed.
Was the reference standard applied regardless of
Two reference standards were used: (1) Culture confirmed
tuberculosis and (2)
the index test result?
Clinical diagnosed tuberculosis. Cases were labelled culture
confirmed if they had cough+fever >2weeks and Mycobacterial
culture positive using Bactec MGIT 960 system. Clinical
diagnosed tuberculosis was defined as cough+fever >2weeks and
two of the following three viz (i) contact with active TB, (ii)
positive tuberculin skin test (undefined) and (iii) Effective
for anti-TB regimen (undefined). Presumably, the reference
standard was applied regardless of the index test result.
However, it should be noted that ‘Culture confirmed’ and
‘Clinical diagnosis’ appear to be mutually exclusive in the
sense that an individual child could have only one of the two.
The authors have not considered a reference standard combining
the two criteria
Was there an independent, blind comparison between the index
test and an appropriate reference (‘gold’) standard of
Culture confirmed TB is an appropriate gold standard. Many
physicians also rely on Clinical Diagnosis as an appropriate
reference, although both have limitations. The authors do
not specify whether the index test and reference tests were
undertaken independently by examiners blinded to the results
Test characteristics and measures
assay vs culture: Sn 64.7%, Sp 70.1%, PPV 15.9%, NPV 95.7%, LR+
2.16, LR- 0.50. Xpert assay vs clinical diagnosis: Sn 46.3%, Sp
98.6%, PPV 98.3%, NPV 51.4%, LR+ 33.1, LR- 0.54 Xpert assay vs
Diagnosis by culture or clinical criteria: Sn 48.5%, Sp 98.6%,
PPV 98.6%, NPV 49.3%, LR+ 33.1, LR- 0.54.
Do the methods described permit replication?
described for smear, culture and Xpert assay are standard,
well-accepted laboratory techniques. The method for collecting
and processing gastric lavage specimens are also appropriate.
Therefore, the methodological aspects in the study can be
applied in the Indian setting. However, the results call for
cautious optimism and further refinements in the test before it
can replace the current reference standards for diagnosis.
LR- = Likelihood ratio of a positive test, LR- = Likelihood
ratio of a negative test, NPV=Negative predictive value. Sn=Sensitivity,
SP=Specificity, PPV=Positive predictive value
On the other hand, the assay appears to diagnose TB
in only about half of those clinically labeled as tuberculosis. This
appears to be a significant advantage in the sense that it could reduce
the burden of unnecessary treatment. However, a noteworthy point is that
the criteria used for ‘clinical diagnosis’ in this study  make it
very difficult to ignore TB, and most physicians would still opt for
treatment (irrespective of the assay results). In that sense, the assay
has not really demonstrated superiority over the clinical diagnosis,
although a negative result appears to rule out infection. Further, among
the 123 clinically diagnosed cases, Pang, et al. have not
provided a detailed break-up of the criteria these cases fulfilled. This
could be important because one of the criteria is ‘effective for anti-TB
regimen.’ Presumably this implies adequate therapeutic response, and can
be determined only several weeks after initial presentation. It would be
interesting to analyze the performance of the Xpert MTB/RIF assay
separately in those diagnosed on this basis, because if the assay
performs well in them, a correct treatment decision (initiation or
withholding) could be made at presentation.
Pang’s analysis of MGIT sensitivity and specificity
against the reference standard of ‘both microbiologically and clinically
diagnosed TB’ is inappropriate because the index test (here MGIT) is
also part of the reference test. Therefore, MGIT could only be compared
against ‘clinically diagnosed TB’.
Another important (but oft-ignored issue) is that the
Xpert MTB/RIF assay does not distinguish between infection and disease,
and also active versus inactive disease (whereas clinical diagnosis
almost always points towards active disease). Likewise culture-based
diagnosis indicates live bacilli (and presumably active disease).
Therefore, it would have been very valuable if Pang’s study had reported
the outcome of children whose therapeutic decision (not to treat) was
based on the assay result despite a clinical diagnosis compatible with
Perhaps the strength of the Xpert MTB/RIF assay lies
in the speed of obtaining results especially with regard to Rifampicin
resistance. For the diagnosis of tuberculosis, speed is of the essence
and a positive test can result in faster initiation of therapy. This
could be particularly useful in smear negative cases, wherein the
theoretical time to diagnosis is shorter than the assay. The Xpert assay
has been developed as a sturdy kit requiring ‘minimal hands-on technical
time’ . This sounds encouraging but can lead to problems associated
with quality control when performed without standardization (as happens
with many molecular diagnostic tests in India today). The superiority of
Xpert assay in terms of rapidity of diagnosing Rifampicin resistance has
to be balanced with the cost and availability at the point-of-care.
It should also be noted that drug resistance detected
by the Xpert MTB/RIF assay is restricted to detection on one (albeit the
most important) gene responsible for it. Of course, Rifampicin
resistance is not synonymous with INH (and thereby multi-drug)
resistance. In such a scenario, the therapeutic regimen for a Rifampicin
resistant case is not fully elucidated .
Extendibility: It would appear that this
study bolsters the WHO recommendation [2, 3] to consider the Xpert
MTB/RIF assay as an alternative to the conventional methods for
diagnosis. On the face of it, the milieu is ripe for introducing a new
test. The clinical setting of Pang’s study (developing country with
relatively low burden of pediatric HIV-associated as well as multi-drug
resistant tuberculosis), the diagnostic challenges, and the current
algorithms for diagnosis are similar to India. Based on this, it is
relatively easy to replicate a similar study in our setting. However,
the data and the study limitations elucidated above suggest that though
we can be optimistic about molecular diagnostic methods, there is a
considerable ground to be covered before the Xpert assay can replace the
current diagnostic methods (despite their relative limitations).
Conclusions: Molecular diagnostic methods such as
the Xpert MTB/RIF assay appear promising, but data do not suggest that
it can be used as a replacement for current diagnostic methods
(smear/culture or clinical diagnosis) in children suspected to have
tuberculosis (not associated with HIV or suspected to be
Joseph L Mathew
Department of Pediatrics,
PGIMER, Chandigarh, India.
Pediatric Pulmonologist’s Viewpoint
After World Health Organization endorsing the
GeneXpert MTB/RIF assay, several studies were conducted on
smear-negative/culture-positive childhood pulmonary and all of them
agreed the advantage of the above tool in the rapid diagnosis of
tuberculosis (TB) and rifampicin-resistant TB . The present study by
Pang, et al.  has concluded that GeneXpert MTB/RIF showed
significantly better performance (sensitivity and specificity of 48.6%
and 98.6%) than MGIT (the sensitivity and specificity 12.1% and 100.0%)
with gastric lavage aspirates (GLA) samples and recommends Xpert may
serve as a useful tool for the diagnosis of childhood TB, especially for
Though MGIT 960 system reports the growth of tubercle
bacilli fast, conventional culture is mandatory to know the drug
resistant status and to formulate an effective regimen. Including
conventional culture in randomized clinical trials can contribute
additional information on this subject.
Though many diagnostic tests for TB have been
identified, most of them are used for research purpose only and a cost
effective rapid diagnostic test is the need of the hour. Rapid tests in
children with suspected tuberculosis would not only improve the
management protocol of the affected child but also allow greater
integration of pediatric tuberculosis into national tuberculosis control
programs. The result of the TB diagnostic test depends both upon the
test and also on the clinical specimen used.
Since children below 6 years are not able to
expectorate the sputum, specimens like gastric lavage aspirate (GLA),
induced sputum (IS), bronchoalveolar lavage (BAL) are used and each one
will have its own advantages and disadvantages. In the present study GLA
samples were used but a trial comparing all the available specimens
against Gene Xpert MTB/RIF add more information. It has been highlighted
that neutralization of GLA sample with sodium bicarbonate would have
inactivated a part of tubercle bacilli. Since PCR based technology can
detect nucleic acids from both dead and live bacilli, neutralization
aspects need a revision. More than this, a delay in the transportation
and storage of GLA samples would have contributed their own share for
the low detection rate of MGIT culture in the present study. Again the
yield of GLA varies widely in various studies (with or without
vancomycin to reduce contamination, with or without nasogastric tube
insitu overnight, frozen against fresh samples, hospitalized versus
ambulatory patient, pulmonary versus adenopathy, extensive versus mild
disease,) emphasizing the fact that GLA technique needs further
Unlike the adult type, pediatric tuberculosis have
many challenges like paucibacillary nature of infection, poor clinical
expression, equal affection of both pulmonary and extra pulmonary system
and difficulty in obtaining good specimens. All these factors make
microbiologic diagnosis of the pediatric TB a difficult task. Lot of
money is wasted on many unnecessary investigations in TB diagnosis and
unfortunately poor and downtrodden are the victims.
WHO play a great role by providing timely information
on useful diagnostic tests and treatment regimen for effective TB
management which is evidenced by its constant recommendation. Abandoning
TB serodiagnosis, promoting Xpert MTB/RIF for smear-negative TB,
simplifying all TB treatment regimens into two categories and
implementing DOTS are the measures which contributed effectively to
reduce the burden of tuberculosis.
Since Xpert assay can determine only Rifampicin
resistance and not resistance to other first and second-line anti-TB
drugs, this fact needs consideration in the evaluation of suspected
resistance. The study concludes by saying that Xpert MTB/RIF assay is an
excellent tool for the diagnosis of smear-negative childhood TB with GLA
samples but our fervent appeal is all precautions should be duly
followed during GLA collection and if the treating physician involves
himself directly, the bacteriologic yield will increase. Since
inadequate specimen is the major drawback in pediatric TB diagnosis,
combining both specimens like GLA and IS taken on the same day, may
further increase the yield.
Madras Medical College,
World Health Organization (WHO) estimated the global
burden of tuberculosis at 8.6 million new cases and 1.3 million deaths
in 2012, with up to 15% burden in pediatric cases . Tuberculosis
(TB) in children has remained relatively neglected mainly due to lack of
sensitive diagnostic methods . However, recent invention of the
Xpert MTB/RIF assay has significantly transformed the diagnostics
algorithm . After its endorsement by WHO, several workers have
started using Xpert MTB/RIF assay for the diagnosis of pediatric
tuberculosis . Nevertheless, very few studies are published on its
utility in smear negative pediatric samples such as gastric aspirates
In this study, Pang, et al.  report that in
smear negative culture positive GA samples, the sensitivity of Xpert
MTB/RIF was 64.7% which is on expected lines. However, the unexpected
finding in this study is high discordance between MGIT culture and Xpert
MTB/Rif results. The study also shows unexpectedly high (29.9%, 58/194)
false positivity of Xpert MTB/Rif in smear and culture negative samples.
We have observed that 92.2% smear and culture negative GA samples will
also be Xpert MTB/RIF negative (concordance) and only 7.8%
bacteriologically negatives samples will be Xpert MTB /RIF positive
(unpublished data). This discrepancy was most likely due to very low
culture yield (8.1%) in their samples, which were probably not truly
negative. Low culture yield was result of harsh treatment given to the
samples, i.e. first neutralized with NaHCO3
and then frozen at –20ºC and
transported to the reference laboratory after 3-4 days. The paper does
not mention that after receiving when these samples were processed for
culture and Xpert MTB/Rif testing by the reference laboratory. Second,
who performed smear microscopy – referral laboratory or source
laboratory? Third, paper is silent on why smear microscopy was not done
from decontaminated samples as a standard protocol? Fourth, how many
cultures were contaminated and how many of these were Xpert MTB/RIF
assay positive? It is presumed that NaHCO3
neutralized samples would have had more chances of contamination in
The authors highlight limitations of their study and
emphasize that Xpert MTB/Rif assay is not ideal for GA samples due to
its low negative predictive value which means overall low (70.1%)
specificity. They also emphasize that NaHCO3
neutralization of GA samples is not advisable if samples have to be
stored and then cultured in MGIT960 system, though these samples may be
used for DNA-based tests, as the DNA of dead bacilli can be amplified by
later methods. The study also shows that 11.6% Xpert MTB/Rif samples
yielded indeterminant Rifampicin-resistance results, which is an
important cost implication.
Department of Laboratory
AIIMS, New Delhi, India
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