Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore.

M.D. MICROBIOLOGY

RAJARAJESHWARI MEDICAL COLLEGE AND HOSPITAL

BANGALORE –74

“COMPARative evAluation OF PERIPHERAL SMEAR EXAMINATION BY LEISHMAN STAIN, FLUoReSCENT STAIN AND IMMUNOCHROMATOGRAPHIC TEST IN DIAGNOSIS OF MALARIA in a teaching hospital”

BY:

Dr. SHALINI S

POST GRADUATE STUDENT,

DEPARTMENT OF MICROBIOLOGY,

RAJARAJESHWARI MEDICAL COLLEGE AND HOSPITAL

BANGALORE –74

Rajiv Gandhi UniveRSITY of Health Sciences, Karnataka, Bangalore

Annexure – II

Proforma for registration of Subjects for dissertation

1 / Name of the candidate
and address ( in block letters) / dR. SHALINI S
POST GRADUATE RESIDENT,
DEPT. OF MICROBIOLOGY,
RAJARAJESWARI MEDICAL COLLEGE AND HOSPITAL, KAMBIPURA, MYSORE ROAD, BANGALORE-74
2 / Name of the Institution / RAJARAJESWARI MEDICAL College AND HOSPITAL, KAMBIPURA, MYSORE ROAD, BANGALORE-74
3 / Course of study and subject / MD MICROBIOLOGY
4 / Date of admission to course / 31may2012
5 / Title of the topic: COMPARative evaluation of THE PERIPHERAL SMEAR examination BY LEISHMAN STAIN, FLUORESCENT STAIN AND IMMUNOCHROMATOGRAPHIC TEST IN DIAGNOSIS OF MALARIA IN A TEACHING HOSPITAL.
6.
7.
8. / BRIEF RESUME OF THE INTENDED WORK
6.1. NEED FOR THE STUDY
Malaria is one of the most serious vector born infection mainly affecting South East Asia, Malaria causes about 1.5-2.7 million deaths every year globally1.
Malaria starts with nonspecific symptoms like fever, headache, body ache, fatigue and abdominal discomfort. Hence, it is difficult to diagnose malaria clinically but the treatment has to be started immediately to avoid complications like black water fever, pernicious malaria2. It is also important to avoid drug resistance in malarial endemic areas due to the over treatment because of non specific nature of the clinical presentation.
The early diagnosis of malaria not only prevents the complications but also reduces the transmission of the parasite in the community3. Therefore precise lab diagnosis and species identification is necessary.
Microscopic examination of blood smears is the widely used method for detection of malaria parasites and remains the gold standard for malaria .But it has a drawback that it is time consuming and requires an expert microscopist and results are poor in cases of low parasitaemia.4
·  Many other methods have been employed for the simple, reliable, and rapid diagnosis of malaria, the most promising of these are the rapid diagnostic test and Quantitative Buffy coat5 and Acridine orange staining.6
·  A major drawback of Rapid Diagnostic Test is more expensive and the sensitivities and specificities are variable7
·  QBC method has a limitation of species identification of the parasites and also has lower sensitivity for detection of exoerythrocytic stages of parasites8,9. So in our study a simple and more efficient method which is a modified method of QBC10 is used to examine malarial parasites in a large drop of blood as wet mount or a thick smear stained with Acridine orange which is a fluorescent stain, it identifies various stages as well as species of malaria parasite.
6.2 REVIEW OF LITERATURE
·  Malaria is one of the most serious vector borne infection mainly affecting tropical and subtropical countries of the world. Malaria causes about 2-5 million deaths every year globally1. Pregnant women and children have an increased susceptibility to malaria with mortality predominant in children 11.
·  Microscopic examination of blood smears is the widely used method for detection of malaria parasites and remains the gold standard for malaria4.But it has a drawback that it is time consuming and requires an expert microscopist and results are poor in cases of low parasitaemia
·  Most new technology for malaria diagnosis incorporates immunochromatographic capture procedure with conjugated monoclonal antibodies providing the indicator of infection. Preferred target antigens are those, which are abundant in all asexual and sexual stages in the parasites. Currently, interest is focused on the detection of Histidine rich protein 2 (HRP2) from Plasmodium falciparum and parasite specific lactate dehydrogenase (PLDH) from the parasite glycolyticpathway found in all species.11,12
·  Binesh Lal. Y et al in their study suggested using two diagnostic tools, antigen detection and smear in conjunction for the early diagnosis of malarial infection or use antigen detection as a primary test as well as a screening tool for obtaining a false positive or negative result and confirming it with gold standard test13
·  DK Mendiratta el al in their study correlated Modified Acridine orange stain with traditional Leishman stain and found out that it is 100% sensitive and specific14.
·  Hemvani et al 6 found Acridine orange (AO) to be better than Leishman stain as they detected 248 cases by AO against 148 by the latter. Acridine orange has the advantage that screening is much faster. However, it requires a fluorescence microscope which is expensive and the AO stained wet mounts cannot be preserved, unlike the Leishman stained smear.
·  Hyun-Hee Kong and Dong-2 Chung in their study concluded that AO method can be applied in mass surveys of malaria since sensitivity and rapidity of diagnosis were high and specificity was also sufficient to differentiate between erythrocytic stages of P.vivax.15
6.3. OBJECTIVES OF THE STUDY
1)  To detect malaria parasite in peripheral blood smear by Leishman stain,
Fluorescent stain and Immunochromatographic test.
2) Comparison of results of all the three techniques.
MATERIAL AND METHODS
7.1 SOURCE OF DATA
The study will be conducted on patients with clinical suspicion of malaria attending Rajarajeswari Medical college and Hospital, Bangalore after obtaining informed consent.
7.2 METHOD OF DATA COLLECTION (including sampling procedure
if any)
A study will be conducted on 100 single venous blood samples based on purposive sampling method collected from clinically suspected cases of malaria infection16 at Rajarajeswari Medical college and Hospital, Bangalore.
·  The blood sample will be screened for malarial parasite by using Leishman stain, Acridine orange stain in peripheral blood smear and HRP – 2antigen, PLDH antigen detection by immunochromatographic method.
SELECTION CRITERIA
a.  Inclusion Criteria
·  Patients of all age groups with fever who are clinically suspected of malaria infection.
b.  Exclusion Criteria
·  Patients with fever due to a proven etiology other than malaria infection (enteric fever, Dengue)
Statistical Analysis
The data collected will be entered in MS Excel sheet. Data will be analyzed using descriptive statistics from SSPS. Sensitivity and specificity of Leishman stain, Acridine O stain and Immunochromatographic test will be compared.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans.
·  yes
·  5ml of blood will be collected by venous puncture under strict aseptic conditions and anticoagulated with EDTA from clinically suspected cases of malaria infection after obtaining informed consent. There will be no financial liability on the patients included in the study.
·  No animal intervention/ investigation will be done.
7.4 Has ethical clearance been obtained from your institution?
Yes
LIST OF REFERENCES
1.  Gogtay NJ, Dalvi SS, Rajgor D, et al. Diagnostic and Prognostic Utilization of Rapid Strip (OptiMAL and Paracheck), Versus Conventional Smear Microscopy in Adult Patients of Acute Uncomplicated P.falciparum malaria in Mumbai, India. J Assoc Physicians India 2003;(51):762-765
2.  White NJ,Breman JG. Malaria and babesiosis disease caused by RBC parasites. In: Harrisons principles of Internal medicine. Kasper DL,et al,editors.16th ed(vol.1):MC.Graw Hill:New Delhi:2005.p.1224-6
3.  Govt of Karnataka:Cicular NO NVBDCP:HE:27:04-05;Ministry OF Health and Family Welfare:2004. p1-4.
4.  WHO. New perspectives:Malaria diagnosis; Approaches to the diagnosis,2000;WHO/MAL.
5.  Pinto MJ, Pereira NF, Rodrigues S,et al (1999) Rapid diagnosis of falciparum malaria by detection of Plasmodium falciparumHRP-2Ag. J Assoc Physicians India 1999; 47: 1076-1078.
6.  Hemwani N, Chitnis DS, Dixit DS, Asolkar MV. Acridine orange stained blood wet mounts for fluorescent detection of malaria. Indian J Pathol Microbiol 1999;43:125–8.
7.  Chayani N, Das B, Sur M, et al. Comparison of parasite lactate dehydrogenase based immunochromatographic antigen detection assay (Opti-MAL) with microscopy for Detection of Malaria Parasite. Indian J Med Microbiolgy2004; 22: 104-106.
8.  Chatnapa Duangdee, Noppadon Tangpukdee, Srivicha Krudsood et al. Use of buffy coat thick films in detecting malaria parasites in patients with negative conventional thick films. Asian Pacific J of Trop Biomedicine(2012) 301-303.
9.  Sing N, Valecha N, Sharma VP Malaria diagnosis by field workers using an immunochromatographic test. Trans R Soc Trop Med Hyg1997; 91: 396-397.
10. Kawamoto H. Rapid diagnosis of malaria by fluorescence microscopy. Lancet 1991:337:624-625.
11. Moody A. Rapid Diagnostic Test for Malaria Parasite. Clin Microbiol 2004; 15 (1): 1-25.
12. Ashley E., McGready R, Proux S et al Malaria. Travel Med Infect Dis2006; 4 (3-4): 159-73.
13. Binesh lal Y, Jayakumar.S ,Kalyani M, et al. Correlation of quantitative Buffy Coat, Blood smear and antigen detection in diagnosing malarial infection. J of clinical and diagnostic research 2011 oct.vol-5(5):961-963.
14. DK Mendiratta,K Bhutada,R.Narang,P Narang.Evaluation of different methods for evaluation for diagnosis of P.Falciparum malaria. IJMM(2006)24(1):49-51.
15. Hyun-Hee Kong , Dong-2 CHUNG. Comparison of Acridine Orange and Giemsa stains for malarial diagnosis. The Korean Journal of Parasitology1999;33(4):391-394.
16. B Shreekanth,Shalini Shenoy M, K.Sai Lella et al. Evaluation of blood smears, Quantitative buffy coat And Rapid Diagnostic tests in Diagnosis of malaria. J Bacteriol Parasitol 2011;2(8).
9. / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF THE GUIDE / Recommended
11. / NAME AND DESIGNATION
(in block letters)
11.1  GUIDE
11.2  SIGNATURE
11.3  CO-GUIDE (if any)
11.4  SIGNATURE
11.5  HEAD OF THE DEPARTMENT
11.6 SIGNATURE / DR D.G SURESH
PROFESSOR
DEPARTMENT OF MICROBIOLOGY
RAJARAJESWARI MEDICAL COLLEGE AND HOSPITAL
DR SANGEETHA.S
HEAD OF THE DEPARTMENT
DEPARTMENT OF MICROBIOLOGY
RAJARAJESWARI MEDICAL COLLEGE AND HOSPITAL
12 / 12.1 REMARKS OF THE CHAIRMAN
AND PRINCIPAL
12.2 SIGNATURE

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