Abstract:
50
clinicoradiologically diagnosed patients of osteoarticular
tuberculosis (dorsal spine n=35, knee n= 8, shoulder n=1, elbow
= 2, psoas abscess with lumbar spine lesion n= 2, lymphadenitis
n=2) were analyzed. Tissue was obtained after decompression
(n=35) in dorsal spine and aspiration (n=15). Direct microscopy
and culture for mycobacteria and other aerobic and anaerobic
bacteria was done in all cases. Histopathological examination (
n=35) and FNAC ( n=15) was done . Polymerase chain reaction
(PCR) using 16srRNA as primer was done in all cases. Serology
was performed by ELISA test (n= 27 dorsal spine) at admission
and one and three months postoperatively. Serum IgG and IgM
levels were measured against 38 kd antigen, A-60 tubercular
antigen respectively.
AFB staining (direct) and AFB culture sensitivity was positive
in 6 (12%) cases. Aerobic / anaerobic culture sensitivity was
negative in all cases. Histology was positive for TB
in 35(100%) and FNAC for (n=15) . PCR was
positive in 49 (98%) cases. All 27(100%) cases were
serologically positive. Serological tests showed fall of IgM
titre and rise of IgG titre at 3 months as compared to values at
admission.
AFB
culture sensitivity is gold standard for diagnosis but takes
longer time and has low sensitivity. PCR is highly sensitive for
detection of tuberculosis (1-2 mycobacteria) but could not able
to differentiate between live and dead bacteria. Serology does
not distinguish between pulmonary and extrapulmonary cases but
the titre correlates well with the disease activity.
J.Orthopaedics 2008;5(1)e10
Keywords:
osteoarticular tuberculosis, PCR, ELISA, AFB culture sensitivity
Introduction:
Tuberculosis is a
chronic infectious disease, primarily affects lungs and by
hematogenous spread can affect other tissues in the body
including bone and joints. Osteoarticular tuberculosis involves
2-5% of all tubercular lesions in the body (1). Out of which 50%
affects the spine (1). The diagnosis of osteoarticular
tuberculosis and in particular tuberculosis spine is
clinico-radiological. However not every patient presents with
the classical picture. Many diseases may mimic tuberculosis
clinicoradiologically. For accurate diagnosis to be established
the tubercular bacteria must be recovered from the lesion (2).
The emerging multidrug resistant strains are posing a threat to
cure the tubercular lesion hence the mycobacterium should be
isolated and subjected to drug susceptibility test (2) In
doubtful cases, the patients are taken for surgery, and the
diagnosis can be established by subjecting the tissue for
histopathological, and bacteriological (AFB stainning and
culture sensitivity) examination. However, histopathology may be
inconclusive, if the patient has already taken antitubercular
treatment for few months. AFB stainning and culture sensitivity
are not always confirmatory, since it being a paucibacillary
disease and the patients are already on antitubercular drugs,
before they are reporting to the hospital, leading to negative
results. Moreover, culture sensitivity takes a longer time,
leading to unnecessary delay in diagnosis and initiation of the
treatment (2). Attempts have been made in the past to develop
serologic methods for detection of antibodies against
mycobacterial antigens. These tests may provide a better
indication of activity of disease. However, these tests have
limitations due to very low antibody titres in many patients on
one hand and significant antibody titres in some healthy
individuals on the other (3,4,5). Newer methods of diagnosis by
molecular methods [polymerase chain reaction (PCR)] have
introduced to partly overcome the problems of traditional
methods. The results can be obtained within 2-3 days, thereby
helping in early diagnosis and treatment (6,7,8,9). Individually
all these diagnostic modalities have varying sensitivity for
diagnosis, which can be increased when they are used in
combination. In the present study an attempt was made in which
clinicoradiological, bacteriological, immunological and
histological observations were correlated for diagnosis of
osteoarticular tuberculosis.
Material and Methods :
The study was conducted in the Department of Orthopaedics,
University College of Medical Sciences & GTB Hospital,
Delhi. 50 cases of osteoarticular tuberculosis (dorsal spine n=
35, knee n= 8, elbow n=2, psoas abscess with lumbar lesion n= 2,
lymphadenitis n= 2) were analyzed. Tissue obtained during
surgery and by FNAC was analyzed for (a) direct microscopy &
culture of mycobacteria. Ziehl-Neelsen stain and
Lowenstein-Jensen media was used for staining and culture of
mycobacteria (10). The positive cultures were identified with a
set of standard proportional tests for species identification
& drug susceptibility testing was performed by proportional
method for streptomycin, isoniazid. Rifampicin, ethambutol &
pyrazinamide. (b) Tissue samples were also analyzed for other
aerobic & anaerobic bacteria. Gram's stain & culture
sensitivity was used for aerobic bacteria. Anaerobic culture
sensitivity was done in Robertson’s cooked meat medium (11).
(c) Histopathologically tissue were stained with hematoxylin
& eosin stain and was seen under microscope for acid fast
bacillus (AFB) & epithelioid cell granuloma, Langherhan’s
cell with or without caseation (12). (d) Polymerase chain
reaction (PCR) for rapid diagnosis of mycobacterium was done
using 16srRNA as a primer, which is a genus specific primer. (e)
ELISA test was used for serological tests (n= 27). Serum IgG
& IgM levels were measured against 38kd & A-60
tubercular antigen respectively. Blood for serology was taken at
the time of admission and then at one and three months
postoperatively for serial rises or fall in immunoglobulin
titres.
Results :
AFB staining (direct) and AFB culture sensitivity was positive
in 6(12%) cases Histopathology was positive for mycobacteria in
all (100%) cases. PCR was positive in 49(98%) cases. All
27(100%) cases were serologically positive. Serological tests
showed fall of IgM and rise of IgG titre at 3 months as compared
to values at admission.
Discussion :
About
30 million people suffer from tuberculosis throughout the world.
About 1-2% of these patients suffers from osteoarticular
tuberculosis. Diagnosis of osteoarticular tuberculosis is
difficult since the organism is fastidious and slow growing (2).
AFB is difficult to isolate in osteoarticular tuberculosis,
since it is a paucibacillary disease and since the lesion is
deep seated; it is difficult to get the tissue (2).
The diagnosis of
osteoarticular tuberculosis in endemic area is
clinico-radiological. It is justified to treat the patients
clinico-radiologically in classical lesions of the bone. The
clinical response can be observed in 4-6 weeks. However, there
would be certain doubtful diagnosis, where procurement of tissue
is required to ascertain diagnosis. In the bone of appendicular
skeleton, tissue may be procured by FNAC or core biopsy. Delay
of few days in the treatment of the limb lesion does not give
rise to severe consequences, as tuberculosis is a slowly
progressive disease. Tuberculosis of the spine is a deep-seated
lesion, which if not diagnosed promptly and treated adequately,
then consequences would be hazardous, as patient may have
grotesque kyphosis and neurological complication (paraplegia).
The typical lesion can be diagnosed and treated
clinico-radiologically with support of newer imaging modalities
like C.T/ MRI, however tissue diagnosis is must, when there is
slightest doubt clinico-radiologically. Tuberculosis of the
spine needs to surgically decompress for certain indication.
Osteoarticular tuberculosis, being a paucibacillary disease and
some of the patients are already on ATT, hence no single
modality like AFB culture sensitivity, AFB staining,
histopathology are capable of ascertaining the diagnosis. The
role of serological tests and PCR (molecular methods) are still
being not defined in osteoarticular tuberculosis. That is why an
attempt is made to evaluate these all-diagnostic modalities to
ascertain their efficacy in establishing the diagnosis. Hence,
we design this study. Sensitivity of AFB staining (direct) in
various series was reported in the range of 25-75%(2). In our
series, we got 8% (4 cases) positivity with direct AFB staining.
Out of these four cases three patients were on ATT for more than
2 months before presentation and one patient was on ATT since 4
days. Out of these four cases in two cases, direct AFB staining
was positive in pus aspirated from the cold abscess. Direct AFB
staining was however negative in the tissue obtained during the
surgery in both of them. No series to the best of our knowledge
had evaluated direct AFB staining as a diagnostic modality in
the diagnosis of tuberculosis of the spine. In pulmonary
tuberculosis, direct AFB staining has an important role in the
treatment of disease, as the conversion of sputum positive to
sputum negative indicates the efficacy of the treatment. However
in osteoarticular tuberculosis, whether the patient is sputum
positive or negative does not indicate the efficacy of the
treatment. In our series, we got 4% (2 cases) positivity with
AFB culture sensitivity. Out of these two cases, one case had
never taken ATT and one case was on ATT since 4 days. Overall,
six (12%) cases were positive for AFB staining & culture
sensitivity. Lakhanpal et al (1976) reported 49.53%
positivity by AFB culture sensitivity (2). Other workers have
reported in a range of 48.6-80% [Dahl 1951,Dobson 1951,Holmdahl
1951,Wilkinson 1953,Weinberg 1957,Hald (Jr) 1964,and Kemp
etal1973, Masood 1992] (2). Lakhanpal et al attributed the lower
percentage to the possible effect of preoperative antitubercular
therapy. Wilkinson also asserted the same point. In the previous
series positivity by AFB, culture sensitivity is higher than in
our series. This may be because more number of patients before
presentation to the hospital is already on ATT for many days.
However 16 (32%) cases in our series had never taken ATT. Out of
these 16 cases only 2 cases were positive for AFB culture
sensitivity. Therefore, this fact, that duration of ATT has any
influence on the positivity of AFB culture sensitivity could not
be substantiated in our study. Factors attributable for less
positivity with AFB Culture sensitivity are paucibacillary
disease (Number of AFB is about103 - 104 /ml), species present
(M. tuberculosis is more likely to be positive than MOTT),
patient already on ATT, Stain used, Observer's experience. The
major limitations of AFB Culture & sensitivity are, it
requires live organisms, long incubation period, low sensitivity
in-patients already on ATT. Although the culture results are not
available for up to four weeks, they prove the diagnosis of
tuberculosis beyond doubt and the opportunity carrying out
sensitivity test adds to its significance (2). Newer rapid
culture techniques for diagnosis of tuberculosis like BACTEC and
BACTEC-alert would be better alternative compared to
conventional methods. In the need of avoiding culture techniques
which has the hazard of handling live organisms various
immunological methods have been developed to detect serum
antibodies against M. tuberculosis. Of all techniques, ELISA is
reported to be more sensitive. The problem of day-to-day
variation and considerable overlap in distribution of antibody
levels in active TB cases and controls were considered as major
hurdles in using ELISA as diagnostic tool alone. Various authors
have tested various antigens, which could be specific for M.
tuberculosis (4,5). Serological positivity in our series was
100%. As per as the current record 16 cases are in chronic
stage, six cases are in chronic active stage, and five cases are
going into chronic stage from the acute stage. There was
significant difference in the values of IgM and IgG at the time
of admission and at 3 months postoperatively. These antibodies
titre did not correlate with the recovery status of the patient,
as patients did not show recovery proportionally to the
declination of IgM titre. IgM titre was diagnostic of activity
of the disease while IgG titre was diagnostic of the chronic
disease. The IgG levels remain high even after full treatment.
Although IgG level have no diagnostic value, it suggests that
the patient has a chronic disease or healed disease. Seven cases
were negative for IgM. Out of these 7 cases 6 cases were on ATT
for more than 2 months and the remaining one had never taken
ATT. Serological tests results did not correlate with the
duration of ATT intake. This means that ELISA values are
dependent on time of taking sample and state or phase of
disease. Whether patient is smear negative or have pulmonary or
extrapulmonary TB cannot be distinguished by this method. These
observations on serological test results are essentially the
same as those in other series [Stroebel et al (1982),
Bhattacharya. A. Et al, (1986)] (4,5). Large number of organisms
required by all the above methods was major limitation in
detection of M. tuberculosis. A single test, which would amplify
the genome, even if single organism were present, was though to
be ideal for detection of paucibacillary TB cases. PCR can
analyze the expression of genes even from single cells. PCR. was
positive in 49 (98%) cases in our series. One case who was PCR
negative had histopathology report suggestive of tubercular
osteomyelitis. Our results with PCR was comparable to other
author series [Noordhock.G.T. (1995), Jatana.S.K. (2000), Van
der Spoel van Dijk A et al (2000)] (6,13,14). A number of target
genes of mycobacteria DNA have been evaluated for diagnosis by
PCR. The most common target used in PCR is IS6110. This sequence
is specific for M.tuberculosis complex and is present upto 20
times in the genome thus offering multiple targets for
amplification. PCR detection of IS6110 in pulmonary and extra
pulmonary tuberculosis, when compared to culture has a
sensitivity, specificity and positive predictability of 83.5%,
99%, 94.2% respectively (15). However IS6110 is a member of IS3
family, the most widely spread group of bacterial insertion
sequences. It is therefore not surprising that sequences
homologous to IS6110 have now also been found in mycobacteria
other than M.tuberculosis complex. This may perhaps account for
the false positive results that were observed in several studies
that used IS6110 as a target sequence that range from
2.3%-20%(15). In addition to being prone to false-positive
results, this target may also yield false-negative results
[40%]. Indeed, several M.tuberculosis strains that lack this
insertion sequence have now been isolated. In the present study,
we have used 16srRNA as target sequence as it is universally
present and hence rules out the chances of false negative
results. It is a genus specific marker, and present in both
typical and atypical mycobacteria. Combination of both PCR
[IS6110 and 16srRNA{multiplex}] is prefered. ADVANTAGES OF PCR
1. It is a highly efficient and rapid method for diagnosis of
the disease [24hr]. 2. A PCR result is of great value in early
diagnosis, particularly in infection of certain body systems
where disease progression is very fast and detection by culture
method is time consuming. 3. As PCR is a very sensitive
technique and could detect as few as 1-2 mycobacteria in the
specimen, treatment may be initiated based on this result, if
clinically indicated. 4. It can differentiate typical and
atypical mycobacteria. 5. PCR requires a very small quantity of
specimen and therefore, even microliters of a fine needle
aspirate can be tested. PCR can detect very low levels of AFB in
the clinical specimens and results are available within 3
days.However misleading results can occur as even the smallest
amount of contaminating DNA can be amplified. PCR test result
needs to be interpreted in its clinical context. A PCR positive
result does not always confirm to culture results. PCR is not a
substitute for culture; it is an addition to the routine battery
of laboratory tests for the rapid and definitive diagnosis of
tuberculosis. DISADVANTAGE not able to differentiate live from
dead organisms, as it is not dependent on bacterial replication.
The positivity of histopathological reports has been reported in
the range of 72-97%(2,16). Histopathological positivity in our
series was 100%. All the cases had histological features
suggestive of tubercular osteomyelitis, confirmed by presence of
caseation necrosis, epithelioid cell granuloma and Langerhans
giant cells. In our series positivity with histopathology was
more as compared to other series [Gardner (1946), Hald .J
(1964)] (2,16). Sometimes the results are inconclusive, as the
patients were already on ATT for many months before reporting to
the hospital. The advantage of histopathology lies in the early
results obtained, enabling the surgeon to embark upon the
appropriate treatment Silverman.Jan.F. Et al (1985) stated that
FNA biopsy could provide diagnostic material for morphologic and
microbiologic confirmation, while avoiding either core biopsies
or open biopsies under general anesthesia. With the
cytomorphologic, recognition of a granulomatous process
presumptive diagnosis of skeletal tuberculosis can be made, thus
expediting early appropriate antitubercular treatment and
excluding other process (17). Mondal .A. (1994) stated that
CT-guided fine needle aspiration cytology is a useful and
minimally invasive method of ascertaining histopathological
diagnosis of vertebral lesions (18). Combined efficacy of
histopathology, culture sensitivity and guinea pig inoculation
as reported by Lakhanpal et al was 100% positive results (2).
Saxena P.S. et al reported the positivity of combined efficacy
as 76.4%(16). Van der Spoel van Dijk A et al (2000) reported
that detection using culture could confirm only three of the 26
clinically diagnosed tuberculosis cases while PCR detection
confirmed disease in 15 cases. The use of PCR increased the
confirmation of clinically probable tuberculosis from 14 using
standard laboratory techniques and histology to 18 of 26 cases.
Calculated sensitivity and specificity for PCR employing culture
as the "gold standard" were 100% (with 95% CI 29.2;
100.0) and 71.4% (55.4; 84.3), which due to low detection
levels, basically excludes culture as a standard for statistical
analysis. Sensitivity and specificity for PCR using histology as
the "gold standard" were 78.6% (49.2; 95.3) and 87.1%
(70.2; 96.4) respectively with positive and negative predictive
values of 73.3% (44.9; 92.2) and 90% (73.5; 97.9) respectively
(14). Only one case (3.7%) was positive for PCR, Histopathology,
AFB staining and AFB culture sensitivity and serological tests
in our series. No series upto best of our knowledge had
evaluated the various diagnostic modalities in tuberculosis of
the spine.
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