evaluation of sensitivity and specificity of csf gene

“EVALUATION OF SENSITIVITY AND SPECIFICITY OF CSF GENE

XPERT AND TO COMPARE IT WITH CSF CULTURE IN

TUBERCULOUS MENINGITS”

DISSERTATION SUBMITTED FOR

M.D GENERAL MEDICINE

BRANCH – I

APRIL 2020

Reg. No: 201711106

THE TAMILNADU

Dr. M.G.R. MEDICAL UNIVERSITY

CHENNAI

CERTIFICATE FROM THE DEAN

This is to certify that this dissertation entitled “ EVALUATION OF

SENSITIVITY AND SPECIFICITY OF CSF GENE XPERT AND TO

COMPARE IT WITH CSF CULTURE IN TUBERCULOUS MENINGITS” is the

bonafide work of Dr. M.DHIVIYA in partial fulfillment of the university

regulations of the Tamil Nadu Dr. M.G.R. Medical University, Chennai, for M.D

General Medicine Branch I examination to be held in April 2020.

Dr. K. VANITHA M.D.D.C.H

THE DEAN,

Madurai Medical College,

Madurai.

CERTIFICATE FROM THE HOD

This is to certify that this dissertation entitled “ EVALUATION OF


COMPARE IT WITH CSF CULTURE IN TUBERCULOUS MENINGITS “is the

bonafide work of Dr. M.DHIVIYA in partial fulfillment of the university

regulations of the Tamil Nadu Dr. M.G.R. Medical University, Chennai, for M.D

General Medicine Branch I examination to be held in April 2020.

DR.M.NATARAJAN MD

Professor and HOD,

Department Of General Medicine,

Government Rajaji Hospital,


Madurai.

CERTIFICATE FROM THE GUIDE

This is to certify that this dissertation entitled “is the bonafide work of

Dr. M.DHIVIYA in partial fulfillment of the university regulations of the Tamil

Nadu Dr. M.G.R. Medical University, Chennai, for M.D General Medicine

Branch I examination to be held in April 2020.

DR.M.NATARAJAN MD

Professor and HOD,

Department Of General Medicine,

Government Rajaji Hospital,


Madurai.

DECLARATION

I, Dr. M.DHIVIYA declare that, I carried out this work on” EVALUATION OF


COMPARE IT WITH CSF CULTURE IN TUBERCULOUS MENINGITS “at the

Department of Medicine, Govt. Rajaji Hospital during the period FEBRAURY

2019 TO JULY 2019. I also declare that this bonafide work or a part of this

work was not submitted by me or any others for any award, degree or diploma to

any other University, Board either in India or abroad.

This dissertation is submitted to The Tamil Nadu Dr. M.G.R. Medical University,

Chennai in partial fulfillment of the rules and regulations for the award of M.D

Degree General Medicine Branch- I; examination to be held in April 2020.

Place : Madurai Dr. M.DHIVYA

Date :

ACKNOWLEDGEMENTS

At the outset, I wish to thank our Dean Dr. K. VANITHA M.D.D.C.H ,

for permitting me to use the facilities of Madurai Medical College and

Government Rajaji Hospital to conduct this study.

My beloved teacher and Head of the Department of Medicine

Prof. Dr.M.NATARAJAN M.D., has always guided me, by example and

valuable words of advice and has encouraged innovative thinking and original

research work done by post graduates.

I shall remain eternally grateful to my beloved teacher, my guide and my unit

chief Prof. Dr.M.NATARAJAN .M.D who has given me his moral support and

encouragement through the conduct of the study and also during my entire

postgraduate course.

I also sincerely thank our beloved professors, Dr.G.Bagialakshmi.M.D.,

Dr.J.Sangumani. MD, Dr.C.Dharmaraj. MD., Dr. David Pradeep Kumar

M.D., Dr. Vivekanandan and Dr.Senthil . MD for their par excellence clinical

teaching and constant support.

I offer my thanks to my unit Assistant Professors Dr. P.S.Vallidevi MD,

Dr. P.Shridharan MD, Dr. Vasanthakalyani MD DCP for their constant

encouragement, timely help and critical suggestions throughout the study and also

for making my stay in the unit informative.

I am extremely thankful to Prof Dr. Prabakaran MD, Head of the department

of Thoracic medicine for their constant support guidance, cooperation and to

complete this study.

I express my thanks to Dr. Brindha, Dr.Santhosh for their help and support in my

dissertation work.

My patients, who form the most integral part of the work, were always kind and

cooperative. I pray to God give them courage and strength to endure their illness,

hope all of them go into complete remission.

I thank my dad, mom, and my sisters who have stood by me during my times of

need. Their help and support have always been invaluable to me.

And last but not the least I would like thank the Lord Almighty for His grace and

blessings without which nothing would have been possible.

CONTENTS

Sl.No Particulars Page No.

1 INTRODUCTION 1

2 AIMS AND OBJECTIVES 6

3 REVIEW OF LITERATURE 7

4 MATERIALS AND METHODS 38

5 OBSERVATION AND RESULTS 49

6 DISCUSSION 71

7 CONCLUSION 78

8 ANNEXURES

Bibliography

Abbreviations

Proforma

Master Chart

Ethical Clearance letter

Anti Plagiarism Certificate

80

84

85

87

88

89

1

INTRODUCTION

“The control of tuberculosis in India was impeded by a

slow, insensitive diagnostic methods, particularly for the detection of

drug-resistant bacilli and in patients with human immunodeficiency virus

infection.

“ After the world health organization (WHO) introduced the gene

Xpert /MTB/RIFin December 2010 for use in TB endemic countries and

declared it a major milestone for global TB diagnosis”.

“The gene Xpert /MTB/RIFtest was a cartridge-based fully

automated CBNAAT (nucleic acid amplification test) for mycobacterium

tuberculosis case detection and rifampicin resistance testing, that was

made especially suitable for use in disease-endemic countries like India

.:” It purifies, concentrates, amplifies (by rapid, real-time PCR) and

identifies targeted nucleic acid sequences in the mycobacterial

tuberculosis bacterial genome ( rpo genome) , and provides results from

unprocessed sputum samples in less than 2 hours, with minimal

manpower .”

2

Tuberculous meningitis (TBM) was one of the most severe forms

of TB. TBM occur when m. Tuberculosis was invaded the membranes

and fluid surrounding the brain and spinal cord. Without appropriate

treatment, the disease will invariably progress, resulting in neurologic

impairment, cns damage and often death. The incidences and prevalances

of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB

in our population are increasing. They require extended treatment and

was associated with higher morbidity and mortality. The risk of

acquiring TB, the rate of progression from latent to active disease, as well

as the morbidity and mortality was associated with TB, all significantly

increase with HIV co-infection . To be added HIV co-infected patients

was five times more likely to develop central nervous system tuberculosis

, which worsens the course of both HIV and TB.

Diagnosing TBM was almost always more difficult than other forms

of bacterial meningitis, partially because subacute onset of symptom

unlike classic bacterial meningitis and because m.tuberculosis is always

paucibacillary in CSF that is difficult to be detected in CSF. When early

diagnosis via detection of m. Tuberculosis in CSF was not possible, an

unconfirmed diagnosis of TBM iwas often made by the combination of

3

classical clinical features and CSF findings. Current diagnostic methods

are mainly culture-dependent (using liquid and/or solid culture media)

and often take more than 6 weeks for the detection of mycobacterium

tuberculosis replication and along with drug sensitivity profile.

Mycobacterial cultures for detection of mycobacterium tuberculosis

was use either solid (lowenstein jensen media) or liquid broth system

(MGIT 960 ). LJ medium is highly specific but was always expensive,

laborious, requires trained personnel, available only in few areas and

takes 6 - 8 weeks to give the results.

Despite culture by MGIT 960 medium gave an earlier results as

compared to LJ medium, delay in diagnosis in smear-negative especially

drug resistant strains was have serious consequences for the patient as

well as the community[7,8]. The geneXpert /MTB/RIF is a molecular

beacon assay for the detection of rifampicin resistance mutations in an 81

bp region of the rpob gene known as rifampicin resistance determining

region ( rrdr).

It was an automated closed-cartridge system used to simultaneously

detect m. Tuberculosis and rifampicin (rif) resistance within 2 hours. It

was also helpful in the semi-quantitative estimation (very low, low,

4

medium and high) of the concentration of TB bacilli in the sample, based

on the cycle threshold. The diagnostic accuracy and effectiveness of the

Xpert MTB/RIF was demonstrated equally in both high resources and

low resources settings. It was dramatically reduced the time of diagnosis

and the time of initiation of therapy thereby reducing the mortality and

morbidity in TBM.

One of the greatest challenges physicians face in modern times with

TBM was making a rapid and accurate diagnosis, which should done as

early as possible using a combination of clinical criteria, radiological

and laboratory diagnostics. Usually, initiation of empiric first-line anti

tuberculous therapy for suspected TBM was often followed to prevent

the mortalitiy and morbidity without laboratory confirmation that TBM

was the correct diagnosis, and without evidence of TB drug-

susceptibility.

In 2011, the world health organization invented geneXpert

/MTB/RIFand fda approved in 2013 for the diagnosis of extra-pulmonary

TB in adults and children. The geneXpert MTB/RIF was simple to

perform , minimal training is required , no cross contamination was seen.

It was just a powersupply and temperature < 30 degree Celsius needed.

5

There was scarce India n data on the sensitivity and specificity of

gene Xpert MTB/RIFin tuberculous meningits patients . India was

ranking first in tuberculosis burden in the world, more data needed to

prove the sensitivity and specificity of gene Xpert MTB/RIF in this

regard . As our institution was an apex centre in south India and being

one of the few centres in the state where geneXpert /MTB/RIFis

available, we were in a better position to generate solid data in this

regard. Hence the purpose of this study.

6

AIMS AND OBJECTIVES

To prospectively determine the sensitivity and specificity of CSF

gene Xpert MTB/RIFin a given set of CSF samples and to compare

it with gold standard culture.

To measure the diagnostic accuracy of gene Xpert MTB/RIFin

CSF culture positive patients.

7

REVIEW OF LITERATURE:

CNS tuberculosis :

Willis in the 17th century, first discovered case of tuberculous

meningitis (TBM) is the most severe form of tuberculosis (TB). As per

the world health Organisation (who) statistics, five countries, namely

India , china,pakistan, Indonesia and south africa, account for over 70%

of the global burden of Disease. Added to it , patients coinfected with

HIV are at more than 20 times Higher risk of developing TB compared

to non‑ infected individuals increasing Their mortality . Although central

nervous system TB accountsfor 5‑ 10% cases of extrapulmonary TB and

only1% of all cases of TB, it is responsible for more Mortality than any

other form of TB, owing to the severity of the disease .

The landmark paper by dastur et al., provided a clearcut

pathophysiology And pathological changes of cns TB, which has been

replaces by Advanced imaging techniques. With the discovery of newer

technologies like Magnetic resonance imaging (mri), more so the newer

8

sequences like Magnetisation transfer (mt), gradient recalled echo (gre),

Susceptibility‑ weighted (sw), diffusion weighted (dw) and Fluid

attenuating inversion recovery (flair) images, the cause And extent of the

disease, its underlying pathophysiology, Its complications, and the

favourable or adverse response to Treatment can now be clearly shown

with almost the same precision During life what was reported in post

mortem samples by Dastur et al.

A careful interpretation of the data provided By dastur et al.,

suggested that majority of their patients had One or more

life‑ threatening complications like Hydrocephalus, infarcts, severe

arachnoiditis, spinal Arachnoiditis and tuberculomas, which correspond

to stage 3 of TB meningits Of medical research council (mrc) staging.

9

Figure 5 a) post‑ mortem brain showing opaque meninges with thick exudates at

base of the brain. (b and c) contrast enhanced computed tomographic scan showing

thick enhancing exudates at base of the brain in basal cisterns(white arrows) in the

same patient. The dilatation of ventricles suggestive of hydrocephalus is shown here.

As almost all the cases studied by dastur et al., had advanced

TBM, especially those in an early stage of TBM. In all these patients,

Magnetization transfer (mt) mr imaging detects the earliest evidence of

Meningitis in the form of hyperintense signal changes on T1 weighted mt

sequences, despite conventional spin Echo sequences show normal study.

The common sites for basal Meningeal enhancement are the

interpeduncular fossa, The pontine, perimesencephalic and suprasellar

cisterns, and The sylvian fissures as shown in the figure 5.

10

The degree of enhancing exudate can vary from a thin layer to a

thick sheet. In advanced cases, these exudates May block the flow of CSF

(cerebrospinal fluid) producing Hydrocephalus. The hydrocephalus can

be either communicating ( mostly) due To basal exudates, or

non‑ communicating either due to narrowing of the cerebral Aqueduct or

obstruction to the CSF flow seen at the level of foramen of lushka and

magendie. With the discovery of newer modalities of imaging, Contrast

enhanced computed tomography (CECT) scan is the imaging modality

of choice for establishing the diagnosis of TBM and MRI provides

much Information about the degree of exudates, degree of

hydrocephalus, the Associated periventricular ooze also about the

development of infarcts and Borderzone encephalitis (BZE).

Figure 6: border‑ zone encephalitis (BZE) affecting the brainstem in a patient with

tuberculous meningitis. (a and b) note hyperintense signal changes (white arrows) on

t2w mr (t2 weighted magnetic resonance) images involving the pons and left

temporal lobe; (c) axial section at the level of pons in the post‑ mortem brain of the

same patient showing diffuse necrosis of almost the entire pons that is suggestive of

bze (white arrow)

11

Border‑ zone encephalitis (bze) occurs in the form of

localised Necrosis of the underlying brain parenchyma due to the

extensive infilrative Exudates . The other complications include gross

compression and narrowing of Large arteries (particularly middle cerebral

arteries) due to meningovasculitis Seen at the base of the brain with

basal ganglionic and thalamic infarction

Complications like optochiasmatic Arachnoiditis as well as spinal

arachnoiditis and tuberculomas are typically Seen with precision on the

contrast enhanced MRI.

CNS tuberculosis includes three clinical categories:

1. TB meningits

2. Intracranial tuberculoma

3. Spinal tuberculosis.

12

All three are encountered with high percentage in regions where

Incidence of TB is high and prevalance of post primary Dissemination is

very high . In regions where incidendce is low Extrapulmonary

manifestations of diseases are seen primarily in Adults with reactivation

disease.

Pathogenesis of cns TB:

During bacillemia , that usually occurs after primary infection or

late Reactivation TB , scattered tuberculous bacilli known as tubercles

are Established in brain, meninges and bones.

The chance of subependymal tubercle with progression and Rupture

into the subarachnoid space is the critical step in the Development if TB

meningitis. Hence meningtis developed as a Progressive primary

infection in infants and children and chronic Reactivation of bacilli in

older adults , with immune deficiency , HIV ,

Drugs (TNF alpha inhibitors):

The spillage of tubercular proteins into the subarachnoid space

Produces an hypersensitive reaction , that progressed to marked

Inflammatory changes seen in the base of the brain .

13

Three features dominating the pathology of cns tuberculosis :

1. Proliferative arachnoiditis:

Mostly seen at the base of the brain , with eventual fibrous

Encasement of cranial nerves and penetrating vessels

2. Vasculitis with resultant aneurysm, thrombosis and infarction of

Vessels located at the base of the brain. Multiple lesions are

Common and variety of strokes syndromes occure .the most

Commonly involved sites are basal ganglia, cerebral cortex, pons

And cerebellum

3. Communicating hydrocephalus :

Due to the extension of inflammatory process to the basilar

Cistern that cause obstruction to CSF circulation and absorption. Less

commonly obstruction to aqueduct can also occur.

Host susceptibility:

The toll like receptor pathway appears to influence the

susceptibility of Humans to TB meningits. This was found in a case

population study design done With 175 HIV patients with TB meningitis

14

185 HIV uninfected populations with Pulmonary tuberculosis, and 392

control patients. A polymorphism in toll Interleukin receptor domain that

contains an adaptor protein which helps in the Signaling from m.

Tuberculous bacteria activated toll like recpetors was Significantly

associated with the susceptibility to meningeal tuberculosis with Odds

ratio 3. The polymorphism is also associated with reduced interleukin 6

Production , that shows immunomodulation is another mechanism of

Susceptibility to TB meningitis .

Clinical features of tuberculous mening its:

It is a subacute meningitis that presents with three discernible phases:

1. Prodromal phase: it usually last for two to three weeks with

insidious

Onset of low grade fever, malaise , lassitude, personality change.

2. Meningitic phase : it is characterised by more neurological features

such

15

As meningimus, protracted headache, vomiting, ,lethargy,

confusion and Varying degrees of cranial nerve palsies and long

tract signs.

3. Paralytic phase : the paralytic phase supervenes as the pace of

illness

Accelerates rapidly, confusion progresses to coma, seizures,

and Hemiparesis and death in some cases. For majority of the

cases, death Occurs within five to eight weeks with the onset of

disease.

About one third of patients have military TB, in which case fundus

often shows Choroid tubercles. It is important to rgecognise the cases

with atypical features. They include acute, rapidly progressive meningitc

symptoms suggestive of Pyogenic meningits or with a slowly progressive

dementia over months or years Characterised by personality changes,

socia withdrawl, loss of libid, memory Deficits. Less common percent of

cases can present with encephalitic course Without overt signs of

meaning its,.

16

Table 1: British Medical Research Council (MRC)

Criteria for assessing disease severity

Stage Features

Stage 1 Non ‑ specific symptoms and signs; no clouding

of Consciousness; no neurological deficits

Stage 2 Lethargy or behavioral changes; meningeal

irritation; minor neurological deficits such as

cranial nerve palsies

Stage 3 Stupor or coma; abnormal movements; seizures;

severe

Deficits such as paresis

Paradoxical reactions also known as pr , an exaceberation of

clinical Signs ( fever , alteration in sensorium , progression of

neurological deficits) Occur after the antituberculous chemotherapy, in

one third of patients with Tuberculousmeningitis. The predictors of

paradoxical reactions include female Gender, HIVcoinfection , shorter

duration of illness.

17

Dignosis of tuberculous meningits :

Early recognition of TB meningitis is always of paramount

importance In our society because the clinical outcome mainly

depend upon the stage At which the therapy is initiated and delay in

treatment Often leads to permanent neurological damage. The diagnosis

of TB meningtis is by CSF examination and radiography.

Spinal fluid examination :

The examination of spinal fluid is of critical for the detection

of TB Meningits. Typically CSF shows elevated proteins and lowered

glucose with Mononuclear pleocytosis. CSF protein ranges from 100 to

500 mg/dl. With Patients in subarachnoid block, it peaks upto 2-6 g/dl, in

association with CSF Xanthochromia that end in grave prognosis. The

CSF glucose is less than 45 Mg/dl in 80 % of cases and usual cell countis

between 100 to 500 cells/ microl. Early in the course of the disease , it is

often atypical with few cells or with Polymorphonuclear leukocyte( pmn)

predominance. It then rapidly changes to Lymphocyte cellular response.

On initiation of antituberculous therapy, CSF Sometimes revert to a

polymorphonuclear cellular reaction, with detioration of Clinical

condition.

18

Measurement of CSF ada adenosine deaminase level may be

Useful for the diagnosis of TBM. However CSF ada is elevated even in

Bacterial meningitis. Also there is clear threshold for the diagnosis. One

meta Analysis include 10 studies in which most an elevated ada is defined

as 10 U/l, estimated the sensitivity and specificity of ada for the diagnosis

79 and 91 percenr respectively.

Hence definitive diagnosis requires demonstration of

tubercle Bacilli in CSF, by smear microscopy or culture. Smear

microscopy is Inexpensive and rapid but insensitive (0–20 %) due to low

microorganism Densities in CSF,while culture techniques are

unacceptably slow which makes It unsuitable as a routine technique for

rapidconfirmatory diagnosis.

Thus, diagnosis of tubercular meningitis should be done in

precise And is based on clinical symptoms, neurologic signs, CSF

findings, ct Scans, and response to anti tuberculous therapy.

Culture and sensitivity :

The importance of CSF culture of m.tuberculosis cannot be

Overemphasized. A large volume of CSF specimens is required for the

culture. In A study of 138 adults with clinical tuberculous meningitis , a

19

bacteriological Diagnosis was achieved in about 82 % of cases , AFB

smear and culture were Found to be positive in 58 and 71 % of cases

respectively. The sensitivity is Improved only following certain

principles :

1: best done in last fluid removed in lumbar puncture and if large volume

is Used

2: can be seen best in smear of the clot.

3: 0.02 ml of the centrifuged deposit should be used in glass slide and

stained by Kinyoun or ziehl neelsen method.

4:between 200- 500 high powered fields should be examineb by more

than one

Observer.

Mycobacterial cultures for detection of mycobacterium

Tuberculosis use either solid (lowenstein jensen media) or liquid broth

system (MGIT 960 ). LJ medium is highly specific but is expensive,

laborious, requires Trained personnel, not widely available and takes 6 - 8

weeks to give the results.

20

Though results by MGIT 960 medium come earlier as compared to

LJ medium, Delay in diagnosis especially drug resistant strains can occur.

CSF specimens should be submitted for nucleic Acids

amplificiation testing whenever possible, particularly in the cases of high

Clinical suspicious and negative AFB staining. In many countries the use

of Xpert MTB/RIF/rif assay as an initial test for the diagnosis of

tuberculous meningitis Including India . In one systematic review and

meta analysis including 18 Studies, the sensitivity and specificity for the

Xpert MTB/RIF/rif assay was found to be 81 and 98 percent respectively

in comparing with the culture. Also to add it is Shown that a high

m.tuberculosis bacterial load measured in pretreatment CSF Using the

Xpert MTB/RIFhas been signifanctly associated with increased disease

severity and inflammatory response and with new neurological events

after therapy

Other nucleic acid amplification test have high specificity but

moderate Sensitivity comparing cultute. In a meta analysis including 63

studies Comprising more than 1300 patients of tuberculous menigitis, the

sensitivity and Specificity of XPERT/MTB/RIF assay were 82 and 99

percent respectively. Nucleic

21

Acid amplification test have the advantage of a rapid results

turnaround time Hence it should be used in combination of smear.

An important point to be considered in use of nucleic acid

amplification test for The diagnosis of TBM is negative predictive value

( NPV) . Test characteristics Of the XPERT/MTB/RIF assay showed

that these tests should be used in confirming With tuberculous meningitis

but cannot be used to rule out tuberculous Meningitis

Xpert/MTB/RIF assay is, however, a simple assay that can be

performed with minimal training. The results are available within two

hours

Cartridge based nucleic acid-based amplification Test(CBNAAT )

or geneXpert /MTB/RIF has been developed as an important tool for

diagnosing TBM. The geneXpert MTB/RIFsystem is a single use

Cartridge based real-time pcr system that performs sample

Decontamination, automated nucleic acid amplification, And

fluorescence-based quantitative pcr. According to recent who guidelines,

Xpert/MTB/RIF should be used as a first method in diagnosis of TBM

than Conventional microscopy and culture in order to reach quick

diagnosis. If Sufficient volume of material is available, concentration

22

methods should be Used to increase yield. The Xpert/MTB/RIF/

detection threshold is Approximately 100 - 130 colony forming units

(cfu)/ml of sample. Comparing

With the culture, -, the detection threshold is <10 cfu/ml For

mycobacterial liquid culture and is >5,000.

23

Background of gene Xpert MTB/RIFssay

Gene Xpert/MTB/RIF assay is one of the best test in modern Times

that is bringing about revolution tuberculosis (TB) control by providing

Rapid diagnosis of tuberculosis disease and simultaneously detecting

drug Resistance. Gene Xpert/MTB/RIF assay has the ability to detect

concurrently the Mycobacterium tuberculosis complex and to detect

whether the given sample is Showing resistance to rifampicin ( RIF ) in

less than 2 hours. When compared

With, standard cultures mycobacterium tuberculosis to be grown a

For atleast 2 to 6 weeks is needed and for conventional drug resistance

tests Further 3 more weeks is needed. Earlier results which are

provided by the Xpert/MTB/RIF assay helps in choosing appropriate

treatment regimens and Arriving at infection control decisions.

The sensitivity of Xpert/MTB/ RIF in a large vietnamese study

was 59.3% [(n = 108/182 (95% confidence interval (ci) 51.8; 66.5)],

whichIs (significantly though only slightly) lower than that of the

Mycobacteria growth indicator tube (MGIT) culture [66.5%(n =

121/182), (95% ci 59.1; 73.3)]. However its specificity Was comparable

24

to that reported for other commercial NAAT [99.5% (95% ci 97.2;

100)].

In study by nguyen thi quynh nhu et al, Xpert /MTB/RIF Was

positive in 108 (59.3%) patients with sensitivity of 59.3% And specificity

99.5%. 4 cases of rif resistance(4/108) was Identified by

XPERT/MTB/RIF.

Patel and colleagues in patna , reported the diagnostic

performance of The gene Xpert MTB/RIFsystem’s Xpert MTB/RIFmTB/

rif assay for the diagnosis of TBM Assay’s overall sensitivity was 62%,

and specificity was 95%..

In the study conducted by sharma kusum et al, multiplex Pcr

was positive in 84.78% cases. The overall sensitivity And specificity was

86.63% and 100 % respectively. In CSF, The pooled sensitivity from

metaanalysis of Xpert/MTB/RIF Compared against culture as a reference

standard was 79.5% (95% ci, 62.0-90.2%) (16 studies, 709 specimens).

Mechanism of gene Xpert MTB/RIFassay:

Gene Xpert/MTB/RIF assay is an automated cartridge based

Nucleic acid amplification test which is usually abbreviated as (cb-naat),

25

That employs a single time use of cartridge with the gene Xpert

MTB/RIFinstrument System. CSF sample is collected from the patient

after lumbar puncture with

Presumptive tuberculosis meningits . CSF sample is thoroughly mixed

with the Sterilirising reagent which is being provided with the assay, and

the cartridge Containing this mixture is placed inside the gene Xpert

MTB/RIF instrument. Automated Processing will be taken over by the

machine itself from this point .

Cartridges that are disposable and are made out of plastic

material It has chambers with multiple slots, with buffers and reagents

that are Preloaded and employed for processing sample, purification

along with DNA Amplification by extraction CSF samples are initially

treated with chemicals

Like sodium hydroxide and sample reagent (sr) containing iso-

propanalol.

Sample reagent (sr) assists in reducing the viability of

mycobacterium Tuberculosis bacilli to ten fold by bringing down the

biohazard related event Risks.

26

After which the treated samples are manually loaded in the cartridge

which Is then provided into the gene Xpert MTB/RIFmachine.

• after washing and purification, then by using ultrasonic

Lysis tuberculosis bacilli are concentrated and then dna within is released

from The sample. Dna which was captured is then amplified using a

Hemi‑ nested rt‑ pcr inside the 192 bp segment of the rpo b gene

• spores of bacillus globigii is used as an internal processing of

samples, And for the control of polymerase chain reaction and this assay

is then adjoined With the assay detecting MTB

Detection of rifampicin resistance:

• “ modern technology named molecular beacon is used for detection of

rifampicin resistance wherein the core region of 81 bp of the rpob gene

Encoding the bacterial rna polymerase is amplified using five different

nucleic Acid hybridization probes .”

The probes that are employed in the molecular beacons are

basically Oligonucleotide probes which have been covalently bonded to

fluorophore at One of the end and a quencher at the other end.

27

“ These oligonucleotide probes bind to the complementary

and the Amplified rpob gene sequence. Five differently labelled probes

which are Specific to the different sequences produced by the wild type

rpob gene Segment are employed. “

• “after binding with the probe , emission of fluorescence light results,

Which indicating sensitivity of the strain to rif. Rifampicin resistance is

Detected by the machine when there is mutation in the segment of rpo b

gene Which results in either getting late to emit fluorescence or by

emitting less Fluorescence from the probes .

Hence cb –naat assay using gene Xpert MTB/RIFtechnology

holds good For diagnosis of tuberculosis in resource poor settings and

high prevalence Countries like India for instant diagnosis and detection

of resistance pattern Against mTB. In addition, as processing of sample,

amplification of DNA by Polymerase chain reaction and along with

detection of resistance to rifampcicn Are integrated in one complete

single unit making it definitely a better and an Attractive, impressive,

precise investigation for the point of care settings across various centres.

28

Advantages of the gene Xpert/MTB/RIF assay

The important advantages of the gene Xpert/MTB/ RIF assay are

that

• quick availability of results, and

• minimal manpower is required to run the test.

In addition to that, the gene Xpert /MTB/RIFassay can identify

Possible case of multidrug-resistant tuberculosis.

MDR TB is one of the form of tuberculosis which is resistant to both

the Effective anti tubercular drug of first line anti tuberculous

chemotherapy Namely, isoniazid (inh) and rifampicin. Rifampicin

resistance is a good Predictor of MDR TB because of resistance to

rifampicin, always co-exists With resistance to the drug isoniazid.

Earlier diagnosis of rifampicin resistance potentially allows

Clinicians to treat patients with tuberculous meningitis to effectively

29

start on Treatment much quicker than awaiting results from various other

types of drug Susceptibility testing available.

For patients who are diagnosed to not have tuberculous meningitis,

rapid Results from the Xpert /MTB/RIFassay may help in contributing to

cost savings By preventing patient from starting on unnecessary treatment

and admission and Most importantly reducing the hazardous

complications of tuberculous Meningitis .

30

Interpretation of gene Xpert MTB/RIFssay:

The gene Xpert /MTB/RIFassay should be interpreted

Concurrently with corroborative clinical, radiographic, and other

laboratory Evidence. Nevertheless the gene Xpert /MTB/RIFassay has

not replaced the Need for smear with microscopy for AFB smear, culture

for mycobacteria, and Drug susceptibility testing based on growth, in

addition to genotyping.. Health Care providers and medical laboratories

should ensure that smples are available For further recommended

mycobacterial testing if needed.

Test results from the gene Xpert /MTB/RIFindicates whether

Mycobacterium tuberculosis bacteria was present in the sample or not .

In some Situations, result is reported “invalid," wherein the test should be

repeated.

If mycobacterium tuberculosis complex was detected, the results

will be displayed along with resistance to rif was

detected

not detected

indeterminate

31

Irrespective of the gene Xpert /MTB/RIFresult, all the patient

specimens Should also be sent for mycobacterial culture( MGIT) to

ensure isolates are Available for testing of drug susceptible strains and

further genotyping.

How to interpret results from gene Xpert MTB/RIF ?

Rif resistance detected

The results that are positive for mycobacterium tuberculosis

complex And for rifampicin resistance means that the sample contains m.

Tuberculosis Bacteria with high probability of resistance to att drug

rifampicin, after that The results should be confirmed by additional

methods of rapid testing. If Rifampicin resistance is confirmed, molecular

testing for drug resistance for both First-line and second-line drugs should

be performed immediately whether XDR Bacilli is present and so it

ensures that an effective treatment regimen can be Initiated as early as

possible for the patients.

32

Rif resistance not detected


complex, But have found to be negative for rifampicin resistance means

that the M.tuberculosis bacteria that is present in the given sample are

probably Susceptible to rifampicin. Nervethless , all the tests that were

reported as Positive for mycobacterium tuberculosis complex should

have growth- based Susceptibility testing to first-line TB drugs.

Rif resistance indeterminate


complex And but if found to have indeterminate status for rifampicin

resistance means That the assay could not accurately determine whether

the tuberculosis bacteria In the CSF are resistant to rifampicin or not .if

so is the result, the resistance to First line TB drugs should be

undertaken based on growth-based susceptibility Method that must be

done concurrently with gene Xpert/MTB/RIF.Staining and automated

acid fast bacilli culture using MGIT -960.

They retrospectively reviewed the records of the CSF samples

in TB Meningitis of one hundred and seventy patients in the period of

one year Starting from january 2016 until november 2016 for AFB stain,

33

AFB culture And geneXpert MTB/RIFassay reports. Sensitivity and

specificity along with PPV and NPV of Xpert MTB/RIFssay and

conventional AFB smear microscopy were Formulated using gold

standard as the liquid culture of0 mycobacterium Tuberculosis.

They found that the among the total 170 patient samples

analysed in The final evaluation and among these, only fourteen samples

were positive by all three methods used in their study. It was calculated

on the basis of analysis the . Overall sensitivity, specificity, PPV and

NPV of Xpert MTB/RIFmTB –rif were 86.8%, 93.1%, 78.5% and 96%

respectively .

By which they concluded that Xpert MTB/RIFmTB-rif assay

recorded better and Peak sensitivity when compared with conventional

smear microscopy for acid Fast bacilli in CSF , and Xpert MTB/RIFmTB

-rif can be an essential tool for prompt And early diagnosis in cases of

high clinical suspicion of presumptive Tuberculous meningitis along

with CSF culture .

They also added that positive Xpert MTB/RIFmTB –rif

results, culture Negative results should be interpreted carefully and

should be well correlated With clinical and past treatment details of the

34

patient, and also agreed on the Well known major benefit of gene Xpert

MTB/RIF that it can also instantly detect Rifampicin resistance which

is especially beneficial in patient having Concomitant multi drug resistant

tuberculosis and HIV associated tuberculosis. .

A recent cochrane review concluded that the Xpert /MTB/RIFhad

Replaced CSF smear showed a pooled sensitivity of 88% (95% credible

Interval [cri], 83 to 92%) and a pooled specificity of 98% (95% cri, 97

to 99%) .

Several studies have reported the usefulness and adavnatages of

the Xpert MTB/RIFmTB/ rif test on extrapulmonary samples, with

overall sensitivities of Over 80% and specificity reaching 100% .

However, the Number of CSF samples in these studies combined was

low, including Only a total of 62 specimens.

Due to the emergent situation in the diagnosis of TB meningits

in Suspected TBM cases and because of a rapid increase in mortality and

Morbidity chances with increase of severity (mortality for grade

patients is Approximately 20%; for grade 3 it reaches 55% [31]), a simple

, easily done Rapid, accurate diagnostic test to detect m.bacilli and to

35

detect rifampin resistance in single step, could have a great impact on

survival.

Venn diagram showing positivity by geneXpert/XPERT/MTB/RIF,

culture, and zn smear microscopy using CSF samples in patients with

definite TBM

Determining the optimal volume of CSF to use and processing

steps to Take in order to improve the sensitivity when using the

geneXpert MTB/RIFis another issue that requires further research. A

study , vadwai et al had previously shown a 33% sensitivity of

geneXpert MTB/RIFagainst CSF m.tuberculosis culture and 29% against

a clinical reference standard (crs).

36

It was suggested that Concentration of CSF by centrifugation ,

and the pellet obtained by Centrigugation is used for processing might

increase the sensitivity.

Another Study used a standard 2 ml of CSF including

concentration of the sample Resulting in a geneXpert

MTB/RIFsensitivity of88% against culture yet only 23% against a CRS .

Recently many publications showed , using various volumes and

Concentration methods tended to demonstrate somewhat higher

sensitivity

Results when comparing geneXpert MTB/RIFagainst culture .

A prospective study done by patel et al ,in south africa found

higher Sensitivity when using 3 ml of centrifuged versus 1 ml of

uncentrifuged Samples (82% vs 47%). A study in vietnam reported 59%

sensitivity using 2 ml or less of centrifuged CSF . A more recent study in

uganda found higher sensitivity when centrifuging

CSF prior to using geneXpert MTB/RIF. It also recommended

collecting a much large Volume of CSF (6–10 ml) to improve the

diagnostic yield.

It is also recommended that if only small volumes of CSF

are available for GeneXpert MTB/RIF then the whole volume may be

37

tested directly in the geneXpert MTB/RIF Cartridge without dilution in

the sample reagent that may also yield high Sensitivity and specificity.

Hence the aim of the present study was to prospectively determine

The diagnostic accuracy of Xpert /MTB/RIFin TB meningitis with

culture .

38

MATERIALS AND METHODS

Setting:

Department of general medicine

Government rajaji hospital

Madurai medical college

Madurai.

Subjects:

This study is conducted in patients admitted with the symptoms

and Signs of tuberculous meningitis in the department of medicine ,

GRH, Madurai during February 2019 to July 2019.

Inclusion criteria:

Patients aged 18 -60 years with symptoms and signs suggestive of

Tuberculous meningitis

Exclusion criteria:

After doing lumbar puncture and CSF analysis

Bacterial meningitis

Viral meningitis

39

Cerebral malaria

Brain abscess

Mixed meningitis

Malignancy.

Sample size :

Based on the sensitivity and specificity of gene Xpert

MTB/RIFin tuberculous meningitis observed in earlier publication and

with 20% allowable error and 99% confidence interval, the estimated

sample size is 60, but i have included 50 patients in our study.

Study design:

This was a prospective and observational study.

Consecutive one hundred tuberculous menigitis patients admitted our

hospital were screened for inclusion and exclusion criteria. Evaluation

plan is summarized in ( fig:1). After enrolling the eligible enrolled

candidates, they were interviewed for their socio-demographic data and

detailed history was obtained, which was followed by clinical

examination. All data thus obtained was recorded in the case form

assigned to the subject.

40

Period of study

February 2019 to July 2019.

Assessment of the patients:

A detailed history about the sym

ptoms and comorbidities was taken from all patients and general

systemic examination was carried out. In patients who had symptoms

suggestive of presumptive TB menigitis based on symptoms, lumbar

puncture is done .CSF is subjected to gene Xpert/XPERT/MTB/RIF( 5

ml) and MGIT culture( 5 ml)

Sensitivity ,specificity, positive predictive value and negative

predictive value with 95 % confidence intervals calculated.the proportion

of positive results for each test ( gene Xpert MTB/RIFand MGIT culture)

is compared using mcnemars test.

41

Evaluation of the patient:

PATIENTS WITH SYMPTOMS AND SIGNS OF TUBERCULOUS MENINGITIS

LUMBAR PUNCTURE DONE

CSF IS SUBJECTED TO GENE XPERT/XPERT/MTB/RIF AND MGIT CULTURE

EVALUATING SENSITIVITY AND SPECITICITY OF CSF GENE XPERT/XPERT/MTB/RIF AND COMPARING IT WITH CSF CULTURE

MEASURING THE DIAGNOSTIC ACCURACY OF CSF GENE XPERT/XPERT/MTB/RIF IN CULTURE POSITIVE PATIENTS .

42

Methods:

With informed consent and under strict aseptic precaution, CSF is

Obtained during standard-of-care lumbar puncture ( figure 1). For the

CSF collected, 2 ml for MGIT processing and 2 ml for

geneXpert/XPERT/MTB/RIF;

The remainder was sent to the microbiology and biochemistry labs

for Additional routine testing.

Figure 1: lumbar puncture

43

MGIT 960 culture

Mycobacteria growth indicator tube (MGIT) is intended for The

detection and recovery of mycobacteria. The MGIT mycobacteria Growth

indicator tube contains 7 ml of modified middlebrook 7h9 Broth base. In

our microbiology laboratory, 2 ml of CSF is Centrifuged 3000 g for 15

minutes. After centrifuging, the Supernatant is discarded and the

precipitate re-suspended in 1 ml Buffer. 0.5 ml of CSF is inoculated an

MGIT tube containing 0.8 ml Of MGIT growth supplement “oadc” +

panta antibiotic mixture (polymyxin b, amphotericin b, nalidixic acid,

trimethoprim, Azlocillin),

After processed specimen is inoculated, MGIT tube is Monitored

either by automated instruments until positive or the end of The testing

protocol.

Tubes are filled with samples in the broth and continuously

incubated At 37°c for 42 days. (fig 2) the tubes are monitored for

increasing Fluorescence to determine if the tube is instrument positive.

44

Figure 2: basic initial steps in MGIT 960 system

This instrument was discovered by becton dickinson (bd) (fig

3). It is specially designed to accommodate mycobacteria Growth

indicator tube (MGIT) and incubate them at 37°c. The Instrument is

deigned in such a way that it scans the MGIT every one Hour for

increased fluorescence. Analysis of the fluorescence is used To determine

if the tube is instrument positive; i.e. Test sample( CSF) Contains viable

organisms. A MGIT positive tube contains Approximately 105 - 106

45

colony-forming units per millilitre (cfu/ml). Culture tubes that remain

negative for a minimum of 42 Days (up to 56 days) and which shows no

signs of positivity Are discarded from the instrument and declared as

negative.

Fig 3: MGIT 960 system

46

GeneXpert MTB/RIFXpert/MTB/RIF:

For geneXpert/XPERT/MTB/RIF, CSF sample was poured into

single use disposable cartridge , which is placed into gene Xpert

MTB/RIFmodule. The results will be obtained within available 2 hours.

Each pcr run consists of an internal control for processing the sample

(DNA extraction) and pcr validity ( presence of inhibitors) with positive

and negative controls being tested everyday. Gene Xpert

MTB/RIFsystem will automatically interpret the results from t

fluorescent signals, with algorithmic calculations encoded in it.

The analyses are in the following categories:

Invalid, if pcr inhibitors are detected with amplification failure;

Negative

Positive. If the report is positive, the strain will be classified as

susceptible or resistant to rifampicin. (fig 4)

47

Figure 4: gene Xpert/XPERT/MTB/RIF cartridge and system SR-

Sterilising reagent

48

Data analysis:

Numerical variables were expressed as mean and standard Deviation

and categorical variables were expressed as frequency and Percentages.

To compare the gene Xpert MTB/RIFand AFB culture mcnemar test is

used .

49

OBSERVATION AND RESULTS

DATA ANALYSIS OF OBSERVATION:

Socio demographic characteristics of the study population

Sex distribution of the study population:

About fifty were included in the study, out of which 72% were

males and the rest 28% were females, which is shown in the table 1. It is

shown that there is high incidence of TB meningits in males thanfemales.

Table 2 : sex distribution of the study population

Sex No. Of patients Percentage

Female 14 28

Male 36 72

Total 50 100

50

36

14

GENDER DISTRIBUTION

Male

Female

51

2. Age distribution of the study population:

Table 2: age distribution of the study population

1413

14

9

0

2

4

6

8

10

12

14

16

< 30 31 - 40 41 - 50 > 50

AGE DISTRIBUTION

No.of cases

52

The age of the patients ranged from 18 to 60 years, with a mean of 45. 91

years, with standard deviation of 15.57.

3. Co morbidities of the population:

1. Type 2 diabetes melitus:

Our study patients when interviewed for comorbidities, only 20 %

of patients gave past history of type-2 diabetes mellitus and the rest 80%

of patients did not report having type -2 diabetes mellitus .

Patients with history of

Type -2 diabetes mellitus

Frequency Percentage

Yes 10 20%

No 40 80%

Age of

patients

Minimum

age in

years

Maximum

Age in

years

Mean age Std. Deviation

18 60 55.91 15.57

53

4. History of pulmonary tuberculosis:

Our study patients when interviewed for comorbidities, around 40

% of patients gave past history of pulmonary tuberculosis and the rest

60 % of patients did not report having pulmonary tuberculosis .

5. History of copd.

Our study patients when interviewed for the history of copd ,

around 20 % of patients gave past history of copd and the rest 80% of

patients did not report having copd.


Copd


Yes 10 20%

No 40 80%


Pulmonary tuberculosis


Yes 20 40%

No 30 60%

54

6. History of hypertension:

Our study patients when interviewed for the history of systemic

hypertension , around 5 % of patients gave past history of hypertension

and the rest 95% of patients did not report having hypertension .


hypertension


Yes 3 5%

No 47 95%

7. History of chronic liver disease: our study patients when interviewed

for the history of chronic liver disease , around 10 % of patients gave

past history of chronic liver disease and the rest 90% of patients did

not report having chronic liver disease .


chronic liver disease


Yes 10 10%

No 40 90%

55

8.History of chronic kidney disease : our study patients when

interviewed for the history of chronic kidney disease , around 10 % of

patients gave past history of chronic kidney disease and the rest 90%

of patients did not report having coronary artery disease.


chronic liver disease


Yes 10 10%

No 40 90%

9.History of coronary artery disease : our study patients when

interviewed for the history of coronary artery disease , around 5 % of

patients gave past history of coronary artery disease and the rest

90% of patients did not report having coronary artery disease


coronary artery disease


Yes 2 5%

No 48 95%

56

0

5

10

15

20

25

30

35

40

20

40

5 5

20

5 5

FREQUENCY OF COMORBIDITIES

57

HIV status of the study population:

Around 5 percentage of HIV individuals has increased

susceptibility for tuberculous meningitis.

Patients reactive for HIV No. Of patients Percentage

Yes 3 5%

No 48 95%

Total 50 100

5%

95%

HIV STATUS OF THE POPULATION

HIV

HIV NON REACTIVE

58

SYMPTOMATOLOGY OF STUDY POPULATION:

Patients had different symptoms like fever, headache, vomiting,

altered Sensorium, seizure, cough, focal neurological deficit and loss of

consciousness. Most common symptoms were fever (90), altered

sensorium (58%), Vomiting (46%).

1. FEVER:

Distribution of patients presenting with fever :

Patients reporting

history of fever


Yes 40 90

No 10 10%

About 90% of cases presented with tuberculous meningits in our

hospital had high grade fever with evening rise in temperature.

59

2. ALTERED SENSORIUM :

Distribution of patients presenting with altered sensorium :

Pts with altered sensorium Frequency Percentage

Yes 39 78

No 11 22%

Around 78 percentage of patients had altered sensorium at the time

of Presentation.

3. HEADACHE AND VOMITING :

Distribution of patients presenting with headache and

vomiting :

Patients reporting

history of headache

and vomiting


Yes 32 65

No 17 35%

Around 65% of patients had features of signs of increased

intracranial Tension with headache, vomiting, with papilledema on

examination.

60

4. SEIZURES

Distribution of patients presenting with seizures :

Patients with seizures Frequency Percentage

Yes 15 30

No 35 70%

5. LOSS OF CONSCIOUSNESS

Distribution of patients presenting with loss of consciousness

Patients reporting

history of loss of

consciousness


Yes 10 5

No 40 9%

61

6. FOCAL NEUROLOGICAL DEFICITS.

Distribution of patients presenting with focal neurological

deficits

Patients reporting

history of seizures


Yes 3 05

No 47 95%

0 10 20 30 40 50 60 70 80 90 100

FEVER

ALTERED SENSORIUM

HEADACHE AND VOMITING

SEIZURES

LOSS OF CONSCIOUSNESS

FOCAL DEFICITS

SYMPTOMATOLOGY OF PATIENTS

62

CHEST X RAY OF PATIENTS IN TUBERCULOUS MENINGITS :

In our study of 50 patients, 39% ( 2o patients )

cases had chest x-ray finding which suggested lesion of pulmonary TB

and 8% ( 4 patients) case had old healed lesion while 53% ( 26 patients)

cases had normal finding.

Serial

no

Chest x ray finding Percentage

1. Pulmonary TB lesion 39% ( 20)

2. Old healed lesion 8%(4)

3. Normal 53%(26)

Lumbar puncture analysis of patients in tuberculous meningitis:

Out of 50 patients in our study , 52% of patients had elevated CSF

protein in the range of 100 to 200 , and 28 % had CSF protein in the

range of 200 -300 and mean CSF protein was. Mean CSF protein level

was 136.5 mg/dl and Standard deviation was 69.58.

63

Out of 50 patients in our study , 65% of patients

had CSF glucose in the range of 40-60. Hence mean CSF glucose is

around 55.6 mg/dl.

0

10

20

30

40

50

60

50-100 100-200 200-300 >300

CSF PROTEIN

PERCENTAGE

Serial no

CSF protein level

(mg/dl)

Percentage of

presentation

1. 50-100 8(4)

2. 100-200 52(26)

3. 200-300 28(14)

4. >300 12(6)

Total 100(50)

64

Out of 50 patients in our study , 55% of patients

had CSF cell count in the range of 50-100 cells/microlitre ( lymphocytic

pleocytosis), 28% had cells in the range of 100- 200 cells /microlitre .

Hence mean CSF cell count is around 68 cells /microlitre.

15

65

182

0

10

20

30

40

50

60

70

20-40 40-60 60-80 >80

PERCENTAGE

Serial no CSF glucose level

(mg/dl)

Percentage of

presentation

1. 20-40 15(8)

2. 40-60 65(32)

3. 60-80 18(9)

4. >80 2(1)

Total 100(50)

65

Serial no CSF cells

(cells/microlitre)

Percentage of

presentation

1. 0-50 5(3)

2. 50-100 55(27)

3. 100-200 28(14)

4. >200 12(6)

Total 100(50)

66

CSF ADA LEVEL:

On interpreting the CSF ADA level 55 % had high ADA levels

10-20 units/litre. With mean CSF ADA level of 15 units/litre.

Serial no CSF ADA

(units /litre)

Percentage of

presentation

1. 0-5 5(3)

2. 5-10 28(14)

3. 10-20 55(27)

4. >20 12(6)

Total 100(50)

67

RESULTS:

Out of 50 patients tested for MGIT culture, 40 patients found to be

positive for culture, for which these samples are tested for drug

susceptibility testing.

Out of 50 patients tested for CSF gene Xpert MTB/RIF, true

positive was found to be 32 for gene Xpert MTB/RIFand false positive

was 4, true negative was 8 and false negative was 8 for gene

Xpert/XPERT/MTB/RIF.

80

20

CULTURE RESULTS

culturepositive

culturenegative

68

RESULTS OF GENE Xpert MTB/RIF

PREVALENCE OF RIFAMPICIN RESISTANCE IN GENE

XPERT MTB/RIFPOSITIVE PATIENTS :

95%

5%

Prevalence of Rifampicin Sensitivity in Gene Xpert/XPERT/MTB/RIF

positive patients

rifampicin sensitive rifampicin resistant

FALSE POSTIVE 4

FALSE NEGATIVE 6

TRUE POSTIVE 32

TRUE NEGATIVE 8

69

OVERALL RESULTS OF CSF GENE XPERT MTB/RIFWITH

RESPECT TO CULTURE :

Comparing CSF gene Xpert MTB/RIFwith CSF MGIT culture,

sensitivity of CSF gene Xpert MTB/RIFwas 84% with 95% confidence

interval between 71.23 and 88.79% specificity was 66.67 % with 95%

confidence interval between 60.00 and 88.17%. The positive predictive

value was 88.89 with 95% confidence intervals between 80.28 and 95.79

% and negative predictive value was 57.14 % with confidence interval

between 27.39 and 66.97%.

Hence with these statistical analysis the gene Xpert MTB/RIFhad

a diagnostic accuracy of 80 with 95% confidence intervals between

65.03 and 89..27%. this proves the gene Xpert MTB/RIFis non inferior

to culture with early and best results making gene Xpert MTB/RIFan

important diagnostic tool for the tuberculous meningitis .

70

“Hence Comparing traditional culture-based methods of CSF

testing, GeneXpert MTB/RIFhad similar yield and best , early

results for both the detection of M. tuberculosis and drug-

susceptibility testing. Including use of the GeneXpert MTB/RIFhad

the capacity to improve the diagnosis and management of TBM

cases in our country.”

84.21

66.67

88.89

57.14

80

0

10

20

30

40

50

60

70

80

90

100

SENSITIVITY SPECIFICITY POSITIVEPREDICTIVE

VALUE

NEGATIVEPREDICTIVE

VALUE

ACCURACY

71

DISCUSSION

The primary aim of our study was to evaluate the sensitivity and

specificity of CSF Gene Xpert MTB/RIFMTB –RIF tuberculosis

meningitis patients and compare it with that of mycobacterial culture

using MGIT – 960 as the gold standard to measure the diagnostic

accuracy of Gene Xpert MTB/RIFMTB RIF.

In this study 50 patients 72 % ( n=36)were male and 28%

patients (n=14) were females. Male to female ratio was 1.2 :1 inciding the

higher incidence of tuberculous meningits in males and mean age of

population was 37.53 years, Most of the patients were in the age group of

less than 3o years and between 40 to 50 years..

Co-morbidities mentioned in our study group were 20% patients

of diabetes mellitus, 5% of hypertension, 40% with past history of

tuberculosis/contact and 20% of COPD, 5% of chronic liver disease, 5 %

of chronic kidney disease. This revealed that higher prevalence of

tuberculosis followed by diabetes in our community presenting with

tuberculous meningitis.

72

“ In Sanches et al study, HIV, DM and cancer were frequent

comorbidities associated with extra-pulmonary tuberculosis and seen in

15.8% (20), 6.3% (8) and 4.8% (6) patients respectively.

DM is a well known risk factor for tuberculosis in our community

because of the following reasons depressed cellular immunity,

malfunction of alveolar macrophages, reduced interferon gamma

,microangiopathy of pulmonary vasculature seen more with diabetic

patients “ .

Relating HIV to tuberculous meniingits, 5 % ( 3 patients had

conifection of HIV nad tuberculous meingitis. “ In a Study by Nathan C

Bahr et al out of 257 patients with meningitis 105(40%) patients were

HIV positive. HIV was considered as major risk factor for tuberculosis.

Patients with HIV and active tuberculosis had increased risk of

extrapulmonary tuberculosis, and this risk was attributed due to reduced

CD4+ count.” In symptomatology, almost all, 90 % patients had high

grade fever, followed by altered sensorium. The least coomon

symptomatology is focal neurological deficits.

73

In our study, analysisng the chest Xray of the patients in

tuberculous meningitis, almost half of the patients in our study 26

patients ( 50%) had normal study and about 20 patients (39%) had active

pulmonary tuberculosis and only 4 patients had old lesion. “In a study

conducted by Sidra Aurangzeb et al out of 100 TBM patients

radiographic findings of pulmonary TB was found only in 30(30%)

patients .The predominant patterns on CXR were apical infiltration

(26.6%), miliary mottling (20%) and hilar enlargement (16.6%).The

relationship between pulmonary and cranial miliary lesions was always

controversial “.

CSF analysis done in our study showed that 52 % patients had

elevated protein in the range of 100-200 with mean value of 136 mg/dl.

And 65% patients had glucose in the range of 40-60 mg/dl wit mean CSF

gluocose of 55.6 mg/dl. Around 55 % patients had lymphocytic

pleocytosis in the range of 50-100 cells/mm3.” In the study conducted by

Kumar K et al, 156 patients had lymphocyte predominance (>50%). High

protein (>45mg/dL) was seen in 173 patients”. cell counts are usually

between 100 and 500 cells/μL. Very early in the disease, neutrophil in

abundance can be seen (ii)high protein levels, usually between 100 and

500 mg/dL, (iii)low glucose, typically less than 45 mg/dL or CSF: plasma

ratio <0.5.

74

CSF ADA level in our patients showed a mean value of 15.3 U/L

(range 10-20 U/Litre).”In the study conducted by Lely solari et al the

validity of CSF ADA in tuberculous menigitis , ADA level >6 U/l had a

sensitivity of 60% and was 94% specific. ADA is released by T cells due

to cell mediated immunity response to the M.tuberculosis . Raised levels

of ADA in CSF was not found to be specific to meningeal inflammatory

disease but it should be used as a test for confirming its cause of menigitis

with high predictive value. CSF ADA level 10 μ/L is very sensitive and

used as a additive investigation to diagnose TBM, especially if the

clinical suspicion is high.

When CSF samples are subjected to MGIT culture, 80% had

culture positive for which drug sensitivity test was added. And 20% had

culture negative. And for GeneXpert MTB/RIFtrue positive was 32 and

false negative was 6 % . The sensitivity of CSF gene Xpert MTB/RIF in

our study was 84.21 and specificity was 66.67 %.The positive

predictive value was 88.89% and negative predictive value was 57.14 %

, with these statistical analysis the gene Xpert MTB/RIFhad a diagnostic

accuracy of 80.00% in diagnosing tuberculous menigitis.” In study by

Nguyen Thi Quynh Nhu et al X-pert MTB/ RIF was positive in 108

(59.3%) patients with sensitivity of 59.3% and specificity 99.5%. 4 cases

of RIF resistance(4/108) was identified by Xpert/XPERT/MTB/RIF.” . “

75

Patel and colleagues reported the diagnostic performance of the Gene

Xpert MTB/RIFsystem’s Xpert MTB/RIFMTB/ RIF assay for the

diagnosis of TBM assay’s overall sensitivity was 62%, and specificity

was 95%”.

76

LIMITATIONS

1. The major limitation of the study is the small number of population

used in study.

2. There is also significant loss of case follow up. The other being

overburden of number of samples (both pulmonary and

extrapulmonary) because the test was done in a tertiary centre. Due

to which there was delay in processing of the sample and hence a

high false negative result.

3. The third limitation was our inability to repeat CSF samples for

comparing different factors such as volume and centrifugation . If

done that could have reduced the false negative result of our study

population.

4. Xpert /MTB/RIFassay was used just an add-on test to culture for

diagnosing TB menigitis and it had not replaced the conventional

methods like smear microscopy, culture, and DST for diagnosis

and management of tuberculosis and for detecting resistance to all

other antituberculous drugs , which is one of the limitations of Gene

77

Xpert MTB/RIFthat made gene Xpert MTB/RIFnot useful in

detecting resistance to other drugs and it cannot be used for

monitoring during the course of treatment.

78

CONCLUSION

This is a prospective study in our tertiary centre so far, to

measure the sensitivity and specificity of gene Xpert MTB/RIFin CSF

samples in patients with tuberculous menigitis .

“The sensitivity of CSF gene Xpert MTB/RIF in our study was

84.21 and specificity was 66.67 %.The positive predictive value was

88.89% and negative predictive value was 57.14 % , with these data

analysis the gene Xpert MTB/RIFhad a diagnostic accuracy of 80.00% in

diagnosing tuberculous menigitis.”

In order to reach a quick diagnosis in tuberculous menigitis , using

CSF specimens, CBNAAT need to be preferentially used as major tool

for the diagnosis and treatment in TBM for reducing the mortality and

morbidity of tuberclulous menigitis .Hence CBNAAT had to be

authenticated in every centres like our tertiary centre where there is high

prevalence of tuberculosis as the test that gives rapid result and also

detects rifampicin resistance in a single step which is the major concern

for every physician with respect to TBM . Clear guidance should be given

79

by WHO regarding CBNAAT use in CSF samples in suspected TBM

patients so that gene Xpert MTB/RIF would play a pivotal role in

diagnosis and treatment of one of the most common medical emergency

in India .

80

REFERENCES

1. Tuberculosis key facts-World Health Organisation, cited at

https://www.who.int

2. Global Tuberculosis report 2018.Geneva :World Health

Organisation 2018

3. Kligman, Stanton, Stgeme, Schor, Paediatric Infectious Disease.

Nelson textbook of pediatrics. Volume 2, Ch 215, pp 1445-1453.

4. Fanning A. Tuberculosis: Extrapulmonary disease. Canadian Med

Asso J. 1999; 160: 1597-603.

5. Iscman MD. Tuberculosis in relation to human immunodeficiency

virus and acquired immunodeficiency syndrome. In: Iseman MD,

editor. A clinician’s guide to tuberculosis. Philadelphia: Lippincott

Williams and Wilkins; 2000 p. 199-252.

6. John M Leonard et al, Central Nervous System Tuberculosis.

Aminoff’s neurology and General medicine, cited at

www.sciencedirect.com, 5th edition, 2014

7. Virendra Kr. Goyal, Approach to Neurotuberculosis, cited at

www.apiindia.org, Ch 24 Medicine updates 2016

8. Sunil Kumar Komanapalli, Uma prasad, Bhagyalakshmi Atla,

Vasundhara nammi et al. Role Of CBNAAT in diagnosing

extrapulmonary tuberculosis in correlation with FNA in a tertiary

https://www.who.int/

81

care centre, International Journal of Research in Medical sciences,

Vol 6

9. Suzuki Y, Nihon Rhinso et al .Tuberculosis infection in the nervous

system. www.ncbi.nlm.nih.gov/m/pubmed/21838041/, 2011 Aug

;69(8):1422-6.

10. Xpert MTB/RIF implementation manual Technical and operational

‘how-to’: practical considerations, 2014, cited at www. who.int

11. Rakesh Bhatia, Dayal R, Jindal S, Agrawal D, Goyal A ; Gene

Xpert for Diagnosis of Tubercular Meningitis. Indian J Pediatr.2016

Nov;83(11):1353-1355 [pubmed]

12. InêsSanches a, Aurora Carvalho, Raquel Duarte Who are the

patients with extra-pulmonary tuberculosis? Rev Port Pneumol.

2015; 21(2):90--93. https://journalpulmonology.org

13. Nathan C Bahr, Lillian Tugume, David R Boulware; Improved

Diagnostic Sensitivity for TB Meningitis with Xpert MTB/RIF of

centrifuged CSF : A Prospective study. Int J Tuberc Lung Dis.2015

Oct;19(10):1209-1215

14. M Modi, Sharma, Prabhakar, Goyal MK, Takkar A et al. Clinical

and radiological predictors of outcome in tubercular meningitis: A

prospective study of 209 patients. Clinical Neurology and

Neurosurgery 161 (2017) 29–34.[pubmed

82

15. Sidra Aurangzeb et al. Chest Radiographic Findings in

Neurotuberculosis without Pulmonary Signs and Symptoms. Cited

at www.jcpsp.com Journal of College of Physicians and Surgeons

Pakistan, 2008; vol.18(1);27-30.

16. Ali Moghtaderi, Niazi A, Alavi-Naini R, Yaghoobi S, Naroule B et

al. Comparative analysis of CSF ADA in Tuberculous and

Nontuberculous meningitis. Clinical Neurology and

Neurosurgery.112(2010) 459-462.[pubmed]

17. Kumar K et al. Diagnosis and treatment of tuberculosis :latest

developments and future priorties. ARH. Cited at

www.arh.amegroups.com/article/view/3820/4592, 22 Aug 2017.

18. Grace E Marx et al. Tuberculous Meningitis: Diagnosis and

Treatment Overview. Tuberculosis Research and Treatment Volume

2011, cited at https://www.hinawi.com/journals/ trt/2011/798764,

Article ID 798764, 9 pages.

19. Lely Solari et al. The Validity of CSF parameters for the diagnosis

of tuberculous meningitis. Int J of Infect Dis. Vol 17:issue 12,Dec

2013; e1111-e1115. https://doi.org/10.1016/j. ijid.2013.06.003

20. Bharat Kumar Gupta et al. Adenosine Deaminase Levels in CSF of

Tuberculous Meningitis Patients. J Clin Med Res. 2010 Oct; 2(5):

220–224. doi:10.4021/jocmr429w

https://doi.org/10.1016/j.%20ijid.2013.06.003

83

21. Gopal Chandra Gosh et al.CSF ADA Determination in Early

Diagnosis of Tuberculous Meningitis in HIV-Infected Patients.

Scientifica Volume 2016. doi:10.1155/2016/5820823.

22. Nguyen ThiQuynhNhu, Dorothee Heemskerk, Do Dang Anh Thu.

Evaluation of GeneXpert MTB/RIF for Diagnosis of Tuberculous

Meningitis. Journal of Clinical Microbiology p. 226– 233 January

2014 .doi:10.1128/JCM.01834-13.

23. Vinod B Patel, Grant Theron, Laura Lenders, Brian Matinyena,

Cathy Connolly, Ravesh Singh. Diagnostic accuracy of quantitative

PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a high burden

setting: A Prospective Study. PLoS Med.2013 Oct.

doi:10.1371/journal.pmed.1001536

24. Sharma Kusum et al. Multiplex PCR for rapid diagnosis of

tuberculous meningitis. J Neurol (2011) 258:1781–1787. J Glob

Infect Dis. DOI:10.4103/0974-777X.112272.

25. Xpert MTB/RIF implementation manual Technical and operational

‘how-to’: practical considerations. www.who.int, SBN: 978 92 4

150670 0 © World Health Organization 2014.

26. UPTODATE medicine.

84

ABBREVIATIONS

TBM-Tubercular Meningitis

CSF-Cerebrospinal Fluid

CBNAAT-Cartridge Based Nucleic Acid Test

WHO-World Health Organisation

HIV-Human Immunodeficiency Virus

MRI-Magnetic Resonance Imaging

AIDS-Acquired Immune Deficiency Syndrome

MDR-TB-Multi Drug Resistant Tuberculosis

ADA-Adenosine Deaminase

CT-Computerised Tomography

PLHA-People living With HIV/AIDS

EPTB-Extra Pulmonary Tuberculosis

MTB/RIF-Mycobacterium Tuberculosis/Rifampicin resistance

RNTCP-Revised National Tuberculosis Control Programme

CFU-Colony Forming Unit

DM-Diabetes Mellitus

CXR-Chest Xray

85

PROFORMA

Name:

Age / Sex:

Occupation:

Presenting complaints:

Past History:

H/o DM, HT, CKD, CVD, DRUG INTAKE, CAD, Thyroid

disorders, Alcohol intake

Clinical Examination:

General Examination:

Consciousness,

Pallor,

Jaundice,

Clubbing,

Lymphadenopathy,

Hydration status

Vitals:

PR

BP

RR

SpO2

86

Systemic examination:

CVS:

RS:

ABDOMEN:

CNS:

Laboratory Investigations

Bio chemistry Microbiology Pathology

RFT HbsAg CBC

LFT HCV

Radiological Investigations :

Chest x ray

CT Brain

evaluation of sensitivity and specificity of csf gene

Documents