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Significance of β-actin gene in Cerebrospinal fluid for the Cellular and Molecular Characterization of Tuberculosis Meningitis
Sharma, Narotam1; Rawat, Anita2; Gupta, Komal3; Agarwal, Ruchi1; Ghanshyam4; Anand4; Ram, Rishi4; Garg, Neelam4; Dhingra, Gulshan K.5
Received: December 20, 2017 Accepted: March 29, 2017 Online: June 30, 2017
Abstract
Tuberculosis meningitis (TBM) is a serious
Central Nervous System infection associated
with significant mortality and morbidity among
the survivors and thus early diagnosis and plays
an important role. The current study signifies
about the clinical relevance of the β-actin gene
in Cerebrospinal fluid in cases with
neurological disorders.
Keywords: Cerebrospinal fluid | Polymerase
chain reaction | Tuberculosis Meningitis | beta
actin gene | latent and active tuberculosis
Introduction
Tuberculous meningitis (TB Meningitis) is a
debilitating form of Central Nervous System
(CNS) tuberculosis with a high morbidity and
mortality in spite of treatment. Though India is
the second-most populous country in the world,
one fourth of the global incident tuberculosis
(TB) cases occur in India annually. Tuberculous
meningitis (TBM) is characterized by a slowly
progressing granulomatous inflammation of the
basal meninges. Individuals with increased risk
for TBM include young children with primary
TB and patients with immunodeficiency caused
by aging, malnutrition, or disorders such as HIV
and cancer (Berenguer et al., 1992; Farer et al.,
1979). Tuberculous meningitis composes of 5-
10% of all tuberculosis cases and is a critical
disease in terms of fatal outcome and permanent
sequel in spite of anti- tuberculosis treatment,
requiring rapid diagnosis and treatment
(Thwaites GE et al., 2002). Many prognostic
factors for TBM have been reported, including
age, the stage of the disease, level of
consciousness, presence of extra-central
nervous system tuberculosis, isolation of
ESSENCE - International Journal for Environmental Rehabilitation and Conservation
Volume VIII: No. 1 2017 [168 – 178] [ISSN 0975 - 6272]
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For correspondence: 1Central Molecular Research Laboratory, Shri Guru Ram Rai Institute of Medical and Health Sciences, SGRR University, Dehradun, Uttarakhand, India 2Uttaranchal P. G College of Biomedical Sciences and Hospital, Dehradun, Uttarakhand, India 3Kanya Gurukul Campus, Gurukul Kangri University, Haridwar,(U.K), India 4Kurukshetra University, Kurukshetra, Haryana, India 5Deptt. of Botany, Pt. L.M. P.G. Collge, Rishikesh, Uttarakhand, India
Sharma et al./Vol. VIII [1] 2017/168 – 178
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Mycobacterium tuberculosis from
cerebrospinal fluid, pathologic biochemical
findings of cerebrospinal fluid (CSF),
hydrocephalus and cerebral infarction (Lu CH
et al., 2001). The diagnosis of TBM is based on
the detection of acid-fast bacilli (AFB) in CSF
smears, and, more commonly, by the isolation
of M tuberculosis from CSF cultures.
Approximately 80 % of the total CSF protein is
derived from the plasma, upon crossing the
blood–brain barrier and another 20 % is secreted
by the CNS (Kroksveen et al., 2011). The
Polymerase Chain Reaction (PCR) is most
sensitive, rapid and a versatile technique and is
increasingly being used in rapid diagnosis of
TB. This study was undertaken to evaluate the
role of TB PCR and significance of β-actin gene
in CSF for the cellular and molecular
characterization of Tuberculous meningitis. We
used primers for beta actin which is a conserved
repetitive element with specificity for M.
tuberculosis complex. The aim of this study was
to compare the efficiency of a PCR with a target
chosen from this cloned fragment with that of a
PCR with the widely used IS6110 sequence in
detecting M. tuberculosis in cerebrospinal fluid
(CSF) samples from patients suspected with
tuberculous meningitis. Materials and Methods:
In this study, a total of 70 CSF samples were
collected from different departments viz.
Medicine, Intensive Care Unit (ICU), Medical
Intensive Care Unit (MICU), High Dependency
Unit (HDU), Pediatrics, Neurosurgery and
Emergency of Shri Mahant Indiresh Hospital,
Dehradun (U.K.) to evaluate the significance of
Beta actin gene in CSF for the cellular and
molecular characterization of patient suspected
of tuberculosis meningitis. All the CSF samples
were processed further for various parameters.
A whole CSF sample was centrifuged at 13,000
rpm for 10 minutes at 4°C. The supernatant was
separated from CSF & then subjected for
nucleic acid extraction. DNA ISOLATION:
Spin column-based nucleic acid purification
was employed for DNA isolation from CSF
specimens. The multiplex PCR was utilized by
the usage of IS6110 gene along with the beta
actin gene (as tabulated in table 5.) as an internal
control, yielding an amplicon size of 123 base
pair for IS6110 and 306 base pair for β-actin
gene.
Gene Sequence (5’ to 3’) Size (bp)
β-actin F TCACCCACACACTGTGCCCATCTACGA 306
β-actin R CAGCGGAACCGCTCATTGCCAATGG 306 Table 1: Oligonucleotide primers used
Results
For the diagnosis of tuberculosis meningitis
samples were subjected to Pathological,
Biochemical, Microbiological, Cellular and
Molecular Profiling of cerebrospinal fluid.
Pathological Profiling
In pathological profiling, RBC, WBC,
Lymphocyte and Neutrophil count were
estimated (Table no. 12). In Pathological
examination RBC, WBC, Lymphocyte and
Neutrophil count were determined, RBC
appeared whenever bleeding was there. Red
cells in CSF signal subarachnoid hemorrhage,
stroke, or traumatic tap. A high WBC count may
indicate infection, inflammation or bleeding.
Lymphocytes should be 40-80%; viral infection
is usually associated with an increase in
lymphocytes counts. In early bacterial
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meningitis the proportion of neutrophils usually
exceeds from their normal range (0-6%).
Biochemical Profiling
In Biochemical examination, Glucose CSF,
Micro-protein CSF and ADA levels were
determined (Table no. 12). Acute bacterial
meningitis is usually associated with a CSF
protein concentration between 100 and 500
mg/dl, CSF protein is almost always high
tuberculous meningitis. A decreased glucose
level is seen in tuberculous meningitis,
Determination of ADA levels in CSF is a rapid
test to distinguish TBM from non- TB
Meningitis especially in Immunocompetent
hosts. Its sensitivity ranges from 75% to 94%
and specificity from 86% to 97%. But the role
of CSF ADA level determination is still
controversial in meningitis patient.
Cellular and Molecular Profiling
In Cellular and Molecular Profiling, DNA from
all the CSF samples was isolated and further
targeted for IS6110 gene, yielding an amplicon
size of 123 base pair and 306 base pair for β-
actin gene. It was seen that out of 70 specimens,
03 came Positive for both the targets i.e., IS6110
gene and β-actin gene, 09 were Positive for
IS6110 gene only and negative forβ-actin gene,
07 were positive for β-actin gene and negative
for IS6110 gene. In CSF examination, Positive
cases for both IS6110 gene and β-actin gene
show different CSF parameter i.e. high level of
RBC in two cases (1 and 70, as shown in Table
no. 12), WBC and lymphocyte in two cases (52
and 70), High level of Glucose in one case (01),
and low level in one case (70), High level of
Micro-protein in all the three cases (1,52,70),
and ADA level in two cases (52,70) were seen.
Positive cases for IS6110 shows high level of
RBC in four cases (10,21,32,43), high level of
WBC in six cases (10,21,26,32,43,61), high
level of Lymphocyte in three cases (8,21,26),
high neutrophils level in four cases
(10,32,43,61), high glucose level in four cases
(8,21,43,61), low level of glucose in two cases
(10,26) high level of micro-protein in six cases
(8,10,16,26,32,43), and high level of ADA in
four cases (10,16,17,26) were seen. Positive
cases for β-actin show high level of RBC in six
cases (34,37,46,48,59,63), WBC in three cases
(37,59 and 67), lymphocytes in all seven cases
(34,37,46,48,59,63,67), high level of glucose in
four cases (34,37,48,63), high level of micro-
protein in three cases (34,37,67) and high level
of ADA in one case (37) were seen.
Gender and Age Wise Analysis
07 Male came positive for both IS6110 and β-
actin gene, whereas 05 Female were positive for
IS6100 gene and 03 Positive for β-actin gene.
Most of the Positivity rate for mycobacterial
infection in CSF cases were in between 21-40
years (06 cases) when IS6110 gene was
amplified and β-actin gene was present in 05
cases in the same age group i.e. in between 21-
40 years of age.
Significance of Beta actin gene: Beta actin gene
can be a very important gene for the diagnosis
of tuberculous meningitis because it shows the
inflammation in brain due to bacterial
infections, the cases which are negative for
IS6110 Gene and positive for Beta actin shows
that there is an inflammation in brain other than
M. Tuberculosis, these includes N.
Meningitides. S. Pneumonia, L.
Monocytogenes, H. Influenza, S.pneumoniae.
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Case No.
Gender/Age Molecular Assay
ZN staining
Result Interpretation β –actin
(306 bp) IS6110 (123 bp)
1 Female/48 years Positive Positive Negative Positive for both IS6110 and β actin gene 2. Female/55 years Negative Negative Negative Negative for both IS6110 and β actin gene3. Female/38 years Negative Negative Negative Negative for both IS6110 and β actin gene 4. Female/21 years Negative Negative Negative Negative for both IS6110 and β actin gene 5. Male/60 years Negative Negative Negative Negative for bothIS6110 and β actin gene 6. Male/22 years Negative Negative Negative Negative for both IS6110 and β actin gene 7. Male/19 years Negative Negative Negative Negative for both IS6110and β actin gene 8. Female/35 years Negative Positive Negative Positive for IS6110and Negative for β actin gene 9. Female/4 days Negative Negative Negative Negative for both IS6110 and β actin gene 10. Male/45 years Negative Positive Negative Positive for IS6110and Negative for β actin gene 11. Female/40 years Negative Negative Negative Negative for both IS6110and β actin gene 12. Male/28 years Negative Negative Negative Negative for both IS6110 and β actin gene 13. Male/41 years Negative Negative Negative Negative for both IS6110and β actin gene 14. Female/1month Negative Negative Negative Negative for bothIS6110 and actin gene15. Female/19 years Negative Negative Negative Negative for both IS6110 and β actin gene 16. Female/45 years Negative Positive Negative Positive for IS6110and negative for β actin 17. Male/23 years Negative Positive Negative Positive for IS6110 and negative for β actin gene 18. Male/14 years Negative Negative Negative Negative for both IS6110 and β actin gene 19. Male/22 years Negative Negative Negative Negative for both IS6110and β actin gene 20. Male/16 years Negative Negative Negative Negative for bothIS6110 and β actin gene 21. Male/40 years Negative Positive Negative Positive for IS6110 and negative for β actin gene 22. Male/62 years Negative Negative Negative Negative for both IS6110 and β actin gene 23. Female/43 years Negative Negative Negative Negative for both IS6110 and β actin gene 24. Male/55 years Negative Negative Negative Negative for both IS6110 and β actin gene 25. Male/28 years Negative Negative Negative Negative for both IS6110 and β actin gene 26. Male/30 years Negative Positive Negative Positive for IS6110 and Negative for β actin gene 27. Female/28 years Negative Negative Negative Negative for both IS6110 and β actin gene 28. Male/60 years Negative Negative Negative Negative for both IS6110 and β actin gene 29. Female/45 years Negative Negative Negative Negative for both IS6110 and β actin gene 30. Male/17 years Negative Negative Negative Negative for both IS6110 and β actin gene 31. Male/58 years Negative Negative Negative Negative for both IS6110 and β actin gene 32. Male/7 years Negative Positive Negative Positive for IS6110 and negative for β actin gene 33. Female/17 years Negative Negative Negative Negative for both IS6110 and β actin gene 34. Female/25 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 35. Female/32 years Negative Negative Negative Negative for both IS6110 and β actin gene 36. Male/28 years Negative Negative Negative Negative for both IS6110 and β actin gene 37. Male/24 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 38. Female/6months Negative Negative Negative Negative for both IS6110 and β actin gene 39. Male/14 years Negative Negative Negative Negative for both IS6110 and β actin gene 40. Male/18 years Negative Negative Negative Negative for both IS6110 and β actin gene 41. Female/24 years Negative Negative Negative Negative for both IS6110 and β actin gene 42. Female/52 years Negative Negative Negative Negative for both IS6110 and β actin gene 43. Female/19 years Negative Positive Negative Positive for IS6110 and negative for β actin gene 44. Male/62 years Negative Negative Negative Negative for both IS6110 and β actin gene 45. Male/48 years Negative Negative Negative Negative for both IS6110 and β actin gene 46. Male/70 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 47. Male/40 years Negative Negative Negative Negative for both IS6110 and β actin gene 48. Male/14 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 49. Female/23 years Negative Negative Negative Negative for both IS6110 and β actin gene 50. Female/23 years Negative Negative Negative Negative for both IS6110 and β actin gene 51. Female/35 years Negative Negative Negative Negative for both IS6110 and β actin gene 52. Female/65 years Positive Positive Negative Positive for both IS6110 and β actin gene 53. Male/8 years Negative Negative Negative Negative for both IS6110 and β actin gene 54. Female/35 years Negative Negative Negative Negative for both IS6110 and β actin gene 55. Male/6 years Negative Negative Negative Negative for both IS6110 and β actin gene 56. Male/57 years Negative Negative Negative Negative for both IS6110 and β actin gene 57. Female/16 years Negative Negative Negative Negative for both IS6110 and β Beta actin gene
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58. Male/22 years Negative Negative Negative Negative for both IS6110 and β actin gene 59. Male/39 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 60. Male/13 years Negative Negative Negative Negative for both IS6110 and β actin gene 61. Male/22 years Negative Positive Negative Positive for IS6110 and Negative for β actin gene 62. Male/08 years Negative Negative Negative Negative for both IS6110 and β actin gene 63. Male/26 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 64. Male/42 years Negative Negative Negative Negative for both IS6110 and β actin gene 65. Male/36 years Negative Negative Negative Negative for both IS6110 and β actin gene 66. Male/9 years Negative Negative Negative Negative for both IS6110 and β actin gene 67. Male/30 years Positive Negative Negative Negative for IS6110 and Positive for β actin gene 68. Male/59 years Negative Negative Negative Negative for both IS6110 and β actin gene 69. Male/66 years Negative Negative Negative Negative for both IS6110 and β actin gene 70. Male/19 years Positive Positive Negative Positive for both IS6110 and β actin gene *bp = Base pair Table 3. Cellular and Molecular Profiling of CSF
Cases
Total number of case for Tuberculosis
meningitis Positive cases for IS6110gene Positive cases for β actin gene
70 12 10 Table 4. Total number of positive cases for IS6110 gene and β actin
gene
Gender Number of cases Positive IS6110 β-actin
Male 44 7 7 Female 26 5 3
Table 5. Gender wise Distribution of Positive cases for Male and Female
Age groups (years) Number of cases Positive for IS6110 Positive for β-actin
0-20 21 2 2 21-40 28 6 5 41-60 16 2 1
Above 60 05 2 2 Table 6: Age wise distribution of Positive cases for IS6110 and β-
actin genes
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A: shows 306 base pair amplicon size for β-actin gene in 1 and 2 well B: shows 306 base pair amplicon size for β-actin gene in 1,2 and 6 well C: shows 306 base pair amplicon size for β-actin gene in 1 well D: shows 306 base pair amplicon size for β-actin gene in 2,6 and 7 Well
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Discussion
Βeta actin is a highly conserved protein
expressed in all eukaryotic cells. It is a stable
cytoskeleton protein which is used as a Loading
control in PCR. Our study was undertaken in
clinical samples of patients from Uttarakhand
and its adjoining states. In our study, 70 CSF
samples were processed, which were with the
history of fever, headache, vomiting, neck
rigidity, altered mental status (i.e. confusion,
coma) and altered sensorium. In CSF
examination, 10 cases out of 70 were positive
for β-actin gene and 12 for IS6110 gene. The
above results show that patient having any
neurological disorder have presence of RBC,
high WBC, lymphocytes, Micro-protein and
low level of Glucose in their CSF. These
parameters show sign of inflammation in
Central nervous system. The PCR for β-actin
gene was most likely to give a positive result
when the CSF sample being tested came from a
patient having any neurological disorder, so it
can be hypothesized thatβ- actin gene can be an
important marker for any neurological disorder
whether it is due to M. Tuberculosis or any other
pathogenic micro-organisms. The presence of
β-actin gene was found in the patients with
neurological disorders other than TBM which is
an important finding as this can be utilized to
differentiate the TBM and other inflammations
of the brain. It could be possible that the
presence of highly specificβ-actin gene in 07
patients which came negative for tuberculosis
meningitis have inflammation of other
pathogenic microorganisms. It could be a
useful tool in supporting diagnosis, in particular
when patients is not suffering from tuberculosis
meningitis but have all symptoms of meningitis,
so presence of beta actin gene can confirm the
presence of other pathogenic micro-organisms
which are responsible for meningitis. It can also
be useful when radiological methods have failed
to establish the presence of pathogenic micro-
organisms in central nervous system.
Interestingly, the 09 patients investigated which
are positive for IS6110 gene comes out negative
for the presence of β-actin gene.
Conclusion
TBM is a serious CNS infection associated with
significant mortality and high morbidity among
the survivors. The only way to reduce mortality
and morbidity is by early diagnosis and timely
recognition of complications following the
progression of the disease and in post-treatment
follow-up.A correlation between disease
severity and PCR positivity could be clinically
useful but, given the variability in the clinical
manifestation of TBM and other neurological
infections need to be evaluated further. The
above protocol for β-actin gene can be further
standardized by multiplexing with IS6110 and
mbp 64 genes for the diagnosis and validation
of internal control for M. Tuberculosis complex.
These methods can be utilized for the
prevalence of strains of M. Tuberculosisin India
and its neighboring countries like Pakistan,
Bhutan, Nepal, and Bangladesh etc. The cases
which were positive for IS6110 gene and
negative for β-actin gene, there can be a
possibility of latent Tuberculosis which can be
further studied by interferon gamma release
assays etc. which will differentiate between
Latent and Active tuberculosis.
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Acknowledgment
The authors are grateful to Honorable
Chairman, Shri Guru Ram Rai Education
Mission for his kind support and guidance.
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