Download RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
PROFORMA FOR REGISTRATION OF SUBJECTS FOR
DISSERTATION
1.
NAME OF THE CANDIDATE DR.DIVYA HARINDRANATH
AND ADDRESS
DEPARTMENT OF MICROBIOLOGY
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES,BANGALORE-70
,
2.
NAME OF THE
KEMPEGOWDA INSTITUTE OF MEDICAL
INSTITUTION
SCIENCES
BANGALORE- 560070
3.
COURSE OF THE STUDY
M.D MICROBIOLOGY
AND SUBJECT
4.
DATE OF ADMISSION TO
31st MAY – 2012
COURSE
5.
TITLE OF THE TOPIC:
“A COMPARISON OF DARK-GROUND MICROSCOPY, BLOODCULTURE,
IgM ELISA AND PCR IN THE DIAGNOSIS OF LEPTOSPIROSIS”
6.
BRIEF RESUME OF THE INTENDED STUDY:
6.1) NEED FOR THE STUDY:
Leptospirosis is a zoonotic disease with a world wide distribution. It has been underdiagnosed and under-reported in India due to lack of awareness of the disease,
inadequate epidemiological data and unavailability of appropriate laboratory diagnostic
facilities.1
The disease symptoms range from a mild flu-like illness to a severe form characterized
by jaundice, renal dysfunction, hemorrhagic diasthesis and multi-organ failure and is
associated with high mortality and morbidity. High index of suspicion is needed in the
diagnosis because it mimics other infectious diseases like malaria, dengue, typhoid and
viral hepatitis.2
Since the clinical features are non-specific, combining clinical expertise and
confirmatory laboratory backup increases the recognition of patients with
leptospirosis.3 But, laboratory tests are complex and diagnostic tests like dark-ground
microscopy, blood culture, IgM ELISA and PCR though available have their own
limitations.
So the present study will compare the sensitivity and specificity of the above tests in
the diagnosis of leptospirosis.
6.2) REVIEW OF LITERATURE:
Leptospirosis is a spirochetal disease caused by the pathogenic members of the genus
Leptospira. Weil, Professor of Medicine at Heidelberg (1886) first described this
disease and whose name has been given to the disease in humans2. Taylor and Goyal
reported the first case of leptospirosis from India in 1929 from Andaman and Nicobar
islands.4
Leptospires are finely coiled filamentous spirochete with hooked or curved ends.
Human infection occurs by direct contact with urine of infected rodent or animal or
from water, soil or vegetable contaminated by urine. The organism can penetrate
abraded skin or intact mucous membrane, after which they enter the circulation and
rapidly disseminate to various tissues. A direct toxic effect of the leptospires has been
proposed to cause vascular damage.2
In tropical and subtropical regions the diseases is endemic and exposure is widespread.
Epidemics are associated with high case fatality(15%) , breakout annually during
heavy rainfall.5 Epidemiological studies indicate that the infection is commonly
associated with farmers, sewage workers and animal handler.2 Study conducted by the
National Reference centre, Regional Medical Research centre, Port Blair in 2000-2001
a seropositivity rate from 0 to 46.8% among all cases of fever was observed from
various parts of India. The positivity rate is highest in south India at 25.6%. It is 8.3% ,
3.5%, 3.1% and 3.3% in northern, western, eastern and central India respectively.2
Leptospirosis has a peak during the monsoon and post-monsoon months, and occurs in
people living in urban slums with poor sanitation and low hygienic conditions.4
Rainfall, flooding and animal infections are the cause of epidemiological break-out.3
A study from jipmer , Puducherry,India showed that IgM ELISA gives rapid result
than other conventional methods with overall positivity of 37%.1 Mathur M etal
reported that IgM ELISA has sensitivity of 100% and specificity of 95.9%, sensitivity
of culture is low because culture are time consuming and require special medium.5
PD Brown etal reported that conventional methods such as dark-ground microscopy
and culture are un-reliable and slow for rapid diagnosis and serology also do not
contribute for early diagnosis, PCR can rapidly detect leptospires in small amount of
clinical samples.6 Sharma etal reported that the sensitivity of ELISA was 79% and that
of dark-ground microscope was 60.5% and a combined efficacy of dark-ground
microscope and IgM ELISA as 96%. Dark-ground microscopy should be tried where
other diagnostic tools are not easily available.7
Timely diagnosis is essential since antibiotic therapy provides the greatest benefit when
initiated early in the course of the disease.1 The present study will assess the efficacy
and compare dark-ground microscope, bloodculture IgM ELISA and PCR in the
diagnosis of leptospirosis.
6.3 OBJECTIVES OF THE STUDY
1. To detect Leptospira in blood using dark-ground microscope
2. To isolate Leptospira in blood culture
3. To assess the efficacy of IgM ELISA and PCR in the diagnosis of leptospirosis
4. To compare the sensitivity and specificity of dark-ground microscope,
bloodculture , IgM ELISA and PCR in the diagnosis of leptospirosis
7
MATERIALS AND METHODS
7.1) SOURCE OF THE DATA
Out-patients and In-patients of KIMS hospital with clinically suspected Leptospirosis
•
PERIOD OF STUDY- one year six months
•
SAMPLE SIZE- 60
•
STUDY DESIGN- comparative study
•
SAMPLING DESIGN- purposive sampling
INCLUSION CRITERIA:
Inpatients presenting to KIMS hospital with Fever,
And any one of the following signs and symptom(as laid down by the
Indian Leptospirosis Society)
•
Jaundice
•
Oliguria
•
Cough, hemoptysis and breathlessness
•
Neck stiffness and altered sensorium
•
Hemorrhagic tendencies including conjuctival suffusion5
EXCLUSION CRITERIA:
•
Patients on antibiotics
•
Patients with autoimmune diseases
•
Diagnosed cases of Typhoid, malaria and Hepatitis2
STATISTICAL ANALYSIS
Data collected will be analysed statistically by computing percentage, mean and
standard deviation.The chi-square test will be used as inferential statistics.Wherever
necessary the results will be depictedin the form of graphs and percentage.
7.2 METHOD OF COLLECTION OF DATA
Sample collection
Patients history taken and consent taken for collection of blood. 5ml of blood drawn by
taking aseptic precautions and transferred into two vials, one containing 1% sodium
oxalate phosphate buffer(for dark-ground microscopy and bloodculture) and the other
dry vial ( for IgM ELISA and PCR)
Dark-ground microscope
The sample is centrifuged at 3000rpm for 5min to sediment the
cellular elements. A drop of plasma is placed on the slide and covered with a cover slip
and examined under high power field of dark-ground microscope. If leptospires are not
seen then plasma is centrifuged at 15000rpm for 30 min and the sediments are
examined by wet film preparation under dark-ground microscope.7
Blood culture
1-2 drops of oxalated blood is inoculated into screw-capped test tube
containing 5ml of EMJH medium. Commercially available Difco enrichment medium
is used to prepare culture media. It is incubated at 300c for 8 weeks. All culture tubes
examined weekly for signs of growth like turbidity. All tubes examined
microscopically by taking a loopful of culture and placed on the slide and covered with
the cover slip and examined under high power objective of dark-ground microscope.8
IgM ELISA
The test is done using panbio LEPTOSPIRA IgM ELISA kit with Leptospira
biflexa serovar, Pantoc antigen, on polystyrene surface of microwell and the test is
performed according to the protocol provided with the kit employing 10 microlitre of
test serum .Plates are incubated for 30 min at 370C. HRP-conjugate anti-human IgM
and the TMB in the kit are used as per the manufactures instructions. Absorbance is
read at 450 nm and readings are interpreted as per the instructions in the kit.The
absorbance of the positive control,negative control and cut-off calibrator, provided in
the kit is used for calculation.8
Polymerase chain reaction(6 ,9)
PCR is done using QIA DNA extraction kits with primers
Lep1- 5’ GGC GGC GCG TCT TAA ACA TG 3’
LEP2- 3’ TTC CCC CCA TTG AGC AAG ATT 5’
The reaction buffer is 10mM of Tris HCL, 50mM KCL, 3mM MgCL2 and 0.1%
gelatin, deoxyribonucleotide triphosphates (dATP,dGTP,dCTP,dTTP) used to a final
concentration of 2.5mM . For each reaction 0.5 U Taq.polymerase is added.
DNA extraction
100microlitre of serum is added to 900microlitre of buffer containing guanidine
thiocyanate and 40microlitre of diatoms.The mixture is vortexed and incubated at room
temperature for 10 min and centrifuged, to pellet ,diatoms complexed with DNA. After
washing with the buffer and 70% ethanol and with acetone, DNA-diatom complex is
dried at 560c for 10min and DNA is eluted in the presence of proteinase K at 560C for
10min.
PCR is carried out in MJ Research thermal cycler and consists of 30 cycles of
consecutive denaturation, annealing of primers and DNA chain extension( 1.5 min at
940C, 1min at55 0C ,2 min at 72 0C) . Amplified products identified as leptospira
specific DNA by visualization of bands of expected size on ethidium bromide stained
agarose gel with DIG labelled oligonucleotide probes. A patient is scored positive if the
serum sample produces an amplicon of 331bp with the above primers.
7.5) DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR
INTERVENTIONS TO BE CONDUCTED IN PATIENTS OR OTHER
HUMANS
•
The venous blood sample will be collected and these tests will be conducted on
the same blood sample.The study does not involve any animal experiment.
7.6) HAS ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION : Yes
8.
List of references
1. Smitha B, shekatkar, Harish BN, Menezes GA, Parija SC. Clinical and
serological evaluation of leptospirosis in Puducherry, India. J Infect Dev Ctries
2010;4(3):139-143
2. Dutta TK, Christopher M. Leptospirosis-An Overview. JAPI 2005:53:545-51
3. Shivakumar S. Leptospirosis-current scenario in India. medicine update 2008;
18:799-809
4. Deodhar D,John M. Leptospirosis: Experience at a tertiary care hospital
In northern India. NatlMed J India 2011;24:78-80
5 Mathur M, De A, Turbadkar D. Leptospirosis outbreak in 2005:L.T.M.G.
Hospital experience. Indian J Med Microbiol 2009;27:153-5
6. Brown PD, Gravekamp C,Carrington DG, Kemp HVD,Hartskeerl RA etal.
Evaluation of the polymerase chain reaction for early diagnosis
of leptospirosis. J,Med Microbiol 1995;43:110-14
7. SharmaKK, Kalawat U. Early diagnosis of leptospirosis by conventional
methods: One-year prospective study. Indian J Pathol Microbiol 2008;51:209-11
8. VelineniS, Asuthkar S, UmabalaP, LakshmiV, SritharanM. Serological
evaluation of leptospirosis in Hyderabad, Andhra Pradesh: A retrospective hospitalbased study. J Med Microbiol 2007;25:24-7.
9. Noubade R, Krishnamurthy GV, Murag S, Venkatesha MD ,Krishnappa G.
Differentiation of pathogenic and saprophytic leptospires by polymerase chain
reaction. Indian J Med Microbiol 2002;20:33-6.
9.
SIGNATURE OF CANDIDATE
10.
11.
REMARKS OF THE GUIDE:
NAME AND DESIGNATION(
IN BLOCK LETTERS) OF
11.1 GUIDE
In recent times leptospirosis has become an
urban problem with significant rate of morbidity
and mortality. Hence this study is beneficial in
the patient management to avoid complications.
Dr. JAGADEESH
PROFESSOR
DEPARTMENT OF MICROBIOLOGY,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES, BANGALORE 70
SIGNATURE
11.2 CO-GUIDE ( if any )
SIGNATURE
11.3
HEAD OF THE
DEPARTMENT
DR. K.L. RAVIKUMAR
PROFESSOR AND HEAD,
DEPARTMENT OF MICROBIOLOGY,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
22nd Cross, 24th Main Rd, Banashankari II
Stage,Banashankari, Bangalore, Karnataka
560070
11.4
SIGNATURE
11.5
REMARKS OF THE
CHAIRMAN & PRINCIPAL
11.6
SIGNATURE
11.7
PRINCIPAL OF THE
INSTITUTTION
DR. M.K. SUDARSHAN
PRINCIPAL AND DEAN,
KEMPEGOWDA INSTITUTE OF MEDICAL
SCIENCES
22nd Cross, 24th Main Rd, Banashankari II
Stage,Banashankari, Bangalore, Karnataka
560070