DISSERTATION REPORT

ON

Seroprevalence studied of Leptospirosis in North India

SUBMITTED TO THE

DEPARTMENT OF MICROBIOLOGY

DR. RAMMANOHAR LOHIA AVADH UNIVERSITY, AYODHYA

IN PARTIAL FULFILMENT FOR THE

DEGREE OF MASTER OF SCIENCE IN MICROBIOLOGY

BY

ANCHAL SINGH

UNDER THE SUPERVISION OF

Dr. Sangram Singh Patel

Department of Microbiology
Sanjay Gandhi Post Graduate Institute of Medical Sciences
Raebareli Road

Lucknow-226014 (India)Date-
 Certificate of HOD

The is to certify that Ms.AnchalSingh, a student of M.Sc. Microbiology, Dr. RamManohar

Lohia Awadh University,Ayodhya has worked in the Serology laboratory. Department of
Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow for her
dissertation from March to November 2021 on the project entitled “Sreoprevalence studied of
Leptospira in North India”. All the data obtained by the candidate herself and the data obtained
is genuine. She has gained practical experience during this tenure and successfully completed her
dissertation work under the guidance and supervision of Dr. Sangram Singh Patel, Department of
Microbiology, SGPGIMS

Dr. UJJALA GHOSHAL

PROFESSOR & HEAD

DEPARTMENT OF MICROBIOLOGY,

SGPGIMS, LUCKNOW-226014,

U.P

Certificate of Guide
This is to certify that Ms.AnchalSingh a student of M.Sc. Microbiology, Dr. Ram Manohar
Lohia Awadh University, Ayodhya has worked in the Mycology laboratory. Department of
Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow for her
dissertation from January to June 2021 on the project entitled “Sreoprevalence studied of
Leptospira in North India”. All the data obtained by the candidate herself and the data obtained
is genuine. She has gained practical experience during this tenure and successfully completed her
dissertation work under the guidance and supervision

Dr. SANGRAM SINGH PATEL

PROFESSOR

DEPARTMENT OF MICROBIOLOGY

SANJAY GANDHI POST GRADUATE INSTITUTE OF MEDICAL SCIENCES,

LUCKNOW-226014,U.P

Dr. Ram Manohar, Lohia, Avadh University, Ayodhya

Centre of Excellence, Department of Microbiology
Dr. Shailendra Kumar

Professor & Head Mobile: 9415077035

Email: shailendrakumar@rmlu.ac.in

Certificate of HOD
The is to certify that Ms.Anchal Singh, a student of M.Sc. Microbiology, Dr. Ram,
Manohar,Lohia Awadh University, Ayodhya has worked in the Serology laboratory. Department
of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow for her
dissertation from March to November 2021 on the project entitled “Sreoprevalence studied of
Leptospira in North India”. All the data obtained by the candidate herself and the data obtained
is genuine. She has gained practical experience during this tenure and successfully completed her
dissertation work under the guidance and supervision of Dr. Sangram Singh Patel, Department of
Microbiology, SGPGIMS.

(Shailendra Kumar)

Declaration
I AnchalSingh certify that the work embodied in this M.Sc. projectSEROPREVALENCE
STUDY OF LEPTOSPIROSIS IN NORTHERN INDIA is my own Bonafede work carried
out by me under the supervision of Dr. Sangram Singh Patel from March to Novemberat
Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences,
Lucknow-226014, UP. The matter embodied in this M.Sc. project has not been submitted for the
award of any other degree/diploma.

I declare that I have faithfully acknowledged, given credit to and referred to the research workers
wherever their works have been cited in the text and the body of this project. I further certify that
I have not willfully lifted up some other’s work, para, text, data, results, etc., reported in the
journals, books, magazines, reports, dissertations, thesis, etc., or available at web sites and
included them in this M.Sc. project and cited as my own work.

Sr. No. Title Page No

Date:
Introduction
Place:
1 1-2

Aims & Objects (ANCHAL

2 3
SINGH)

Review of Literature
3 4-16

Material & Methods

4 17-29

Results
5 30-35

Discussion
6 36

Conclusion
7 37

References
8 38-40
 LIST OF ABBREVIATION

ELISA Enzyme - Linked Immunosorbent Assay

MAT Microscopic Agglutination test

EMJH Medium Ellinghusen-Mccullough Johnson- H

IgM Immunoglobulin M

TMB Tetramethylbenzidine

HRP Horseradish peroxidase

IgG Immunoglobulin G

PC Positive controle
 NC Negative controle

RPM Revolution per miter

OD Optical density

OPD Out Patient Department

IPD In Patient Department

ACKNOWLEDGMENTS

The Acknowledgment expression is an effort to produce my overwhelming sense of gratitude

devotion and regards to those entire persons who helped during my dissertation work. I take this

opportunity to express my deep gratitude and appreciation to the people who helped directly and

indirectly to compose my work.

It is great pleasure for me to knowledge my thanks to Dr. Ujjala Ghoshal Professor and Head

Department of Microbiology. SGPGIMS Lucknow, for granting me permission to join as a

dissertation trainee in the institute.

I do not have words of appreciation and inclination to return kindness of my esteemed mentor

Dr. Sangram Singh Patel, Department of Microbiology, SGPGIMS Lucknow for

hisencouragement, devoting his valuable time and ever helping attitude imparted to me form
beginning till the last moment of this work. It is only her blessing, masterly which help me

complete this work.

I also take this opportunity to express a deep sense of gratitude to Dr. Pradeep Paul (Senior

Resident) for this ceaseless encouragement, cordial support, valuable information and guidance.

His continuous attention, motivation and deep interest throughout the tenure of my work

possible.

I am also thankful to all of the Department of Microbiology for their continuous support. I am

very thankful to Mr. Virendra Kumar Yadav and Mr. Ram Kumar Pal laboratory staff of

department of microbiology.

Who have been a source of constant help and encouragement during my Dissertation?

I am indebted to Dr. Shailendra Kumar Professor Head of Department of Microbiology, Dr. Ram

Manohar LohiaAwadh University Ayodhya for this support all through my Master degree and

special thanks to my teachers.

Dr. Rajeev Gaur, Dr. TuhinaVerma, Dr. Ranjan Singh Dr. Anurag Singh and Dr.

ManikantTripathi.

At last, I would like to thanks all the patients who have unknowing been the basic foundation of

the dissertation

Above all I am thankful to almighty god for making me wort while to conduct and complete this

study for giving me the strength and vigor to proceed in my life.

Your sincerely
 ANCHALSINGH

Introduction

Traditionally, Leptospira has been the most overlooked tropical disease. It’s a zoonotic disease

spread by Leptospiraspirocheates. (1,2).Leptospirais divided into two species with different

metabolic properties; L. interrogans which includes pathogenic strain and L.biflexa, including

saprophyte strain isolated from the environment. 3,4}. All animal pathogenic serovars can also

be pathogenic to humans, transmission to humans occurs through penetration of the organism

into the blood stream via cuts, skin abrasions or mucus membranes. {5} The increased

availability of Next-generation sequencing (NGS) has also allowed for the discovery of new

Leptospira species. We were also able to identify a dichotomy in these pathogenic species which

was support by other genetic features. This research will advance various elements of

Leptospirosis research including diagnosis and basic information as well as the evolution of
species ecology and virulence {6,7} Before it wasidentifying the disease was known by a variety

of names in different parts of the world. It was dubbed “Autumn fever” or “seven-day fever” by

some and schlammafieber (mud fever) by others. {8,9}

Weils illness is the popular name for general Leptospirosis. Cancola fever 7 days fever, hervest

fever, field fever, rat catchers,

3R associated with Leptospira

R – Rainfall

R – Rice field

R – Rodent

Blood urine, CSF, infected tissue, body fluids can be used for the diagnosis of Leptospiral

infection. The diagnosis of Leptospirais challenging and cumbersome due to various reason like

not all endemic areas are provided with a laboratory setup due to requirement of a sophisticated

laband well-trainedlaboratory personnel, and the infection mimics other infectious agents like

hepatitis, dengue, scrub typhus.

MAT- (MicroscopicAgglutination Test)

The Microscopic Agglutination Test(MAT) is gold strand for serodiagnosis of Leptospirosis

because of its unsurpassed diagnosis specificity, it uses panicle of live Leptospirosis, ideally

recent isolate, presently the circulating serovars from the area where the patient became infected.

The most common way to diagnose Leptospirosis is through serological tests either the
Microscopic Agglutination test (MAT) which detect serovar- specific antibodies, or a solid phase

assay for the detection of IgM antibodies.

ELISA- (ENZYME LINKED IMMUNOSORBENT ASSAY)

Enzyme is used to label one of the components of immunoassay (e.g.Antigen and

Antibody).ELISA is the method of choice in big laboratories as large number of samplescan be

tested together using the 96 wells microtiter plate. But it is not preferred in small laboratories. It

is a economical takes 2-3 hours (rapid test takes 10- 20 minutes)expensive ELISA is the

mostsensitive immunoassay, thus is the preferred screening test at blood banks at tertiary care

sites. Its specificity used to be low but now with use of purified recombination and synthetic

antigens, and monoclonal antibodies, ELISA has become more specific. It needs equipment such

as ELSIAwasher andreader {9)

AIM & OBJECTIVE

AIM – To evaluated the Seroprevalence pf Leptospirosis among patients admitted to tertiary

care hospital.

Objectives -

1- To estimate the Seroprevalence of Leptospirosis among patients admitted to \ received

fromtertiary care hospital.

2- To assess the risk factors associated Leptospirosis.

3- To find out the correlation between Leptospirosis and other tropical infections during the

study periods.
 REVIW OF LITRATURE

NTRODUCTION

Leptospirosis, one of the most widespread zoonotic disease with high morbidity and mortality, is

caused by pathogenic Leptospira species. The bacteria are hosted in animal kidneys formonths

and even years and the bacteria are released in the environment by the urine of these animals.

The transmission to humans occurs by contact with infected animal or contaminated

waterLeptospira are thin highly motile pathogenic bacteria that are best visualized by dark

ground microscopy. Although these bacteria are not stained by the grams stain, the Fontana Stain

which is a silver impregnation staining method we can used successfully of electron microscope.
Leptospirosis encompasses a wide spectrum of clinical and subclinical disease in both human

and animal. Rats and other rodents are the most important sources. Livestock farming plays an

important role as a major occupational risk factor for human leptospirosis and formed deer is one

of the contributing factors.{10} The bacterium of Leptospira can enters the body via the mucosal

membrane of the eyes, nose, throat, or via cuts or abrasions in the skin. Leptospirosis is

associated with low hygiene levels such as inadequate sanitation and polluted muds and waters in

human habitations. These condition of risk factor for leptospirosis that their association with

disease has been confirmed previously.

The prevalence and epidemiology of leptospirosis were investigated through many studies

worldwide. Differences in seroprevalence and infection rats of Leptospirosis strongly driven by

environmental factors including high rainfall, flooding, natural disasters, population growth

urbanization and poor sanitation and hygiene, Also, these rats are depending on individual

behavior including swimming in fresh water, working outdoor and contact with domestic and

wildlife animals. The specific EMJH (EslinghausenMc Cullough Johnson-Harris) medium use

for Leptospirosisand the best visualised by dark field electron microscope. And using stainfor

leptospirosis FONTANA Stain which is a silver impregnation staining. The Golgi silver

impregnation technique relies on chemical preparation of thick blocks of tissue after which

grains of metallic silver crystallize inside the membranes of individual cells, producing a dense

black precipitate that highlights every detail of the cell body and dendrites against a golden

background.

BIOLOGY OF LEPTOSPIRA
The development of Leptospira necessitated spicy circumstances. Pathogenic Leptospira in the

environment are dependent on various conditions, including pH, temperature, and the presence

of inhibitory compounds, according to stream river organs on liver tissue and dead animals, as

well as dilute milk.{11}Leptospira in room temperature water under laboratory conditions. At

pH7.2 to 8.0last several months.{12} Leptospira spp. Is a genus of parasitic worms that live in

the soil outside the host, don’t multiply they demand high humidity for greater than 50c in the

environment and while they can survive for severalmonths in the lab, they are not suitable for

use with river water. They thrive in nutrient- rich condition including vitamins, long chain fatty

acids and ammonium salt.{13}

Fig.1 –Leptospirainterrogans
 Fig 2-Leptospira magnified in dark field microscope

MORPHOLOGY

There are corkscrew-shaped bacteria that differ from other spirochaetes belonging to the

Leptospira family Leptospirosis and the genus Leptospira.Leptospira are motile, and their bodies

are tiny in diameter, necessitating examination with dark field microscopy or phase contrast.

This pH 7.8.

Cell Structure of Leptospira

Leptospira are thin and spiral-shaped with long hair like structure. It has unique hooked ends on

both sides which differentiated Leptospira from other Spirochaetes. The average diameter of

Leptospira is approximately 0.15µm; the length ranges between 6 and 20µm. they motile with
periplasmicflagellum endo which is responsible for its distinctive cork-screw motile form other

spirochaetes, which also contributes to pathogenicity {14} Leptospiraspecies exhibit increased

swimming speeds under condition of high viscosity {15}

Due to their delicate structure make, they are best seen under dark- field microscopy without

staining. Leptospira are gram variable, mostly appear gram negative, and also possess properties

of gram positive bacteria. Unlike other major spirochete genera such as Treponema or

Borrelia,Leptospira possesses lipopolysaccharides(LPS) on its surface, which also contributes to

pathogenicity. The candidate of virulence factors in Leptospira that might contribute to the

infection including outer membrane proteins, hemolysins, lipopolysaccride and other surface

proteins, as well as adhesion molecules {16} flagella-The Leptospira discovered two

periplasmic flagella with polar insertion in the periplasmic region that are responsible for

motility; the flagella sheath and core are made up of the FlaA and flab protein

respectively.Electron microscopy revealed that flaB mutant lacked Endo flagella was nonmotile.

Life cycle of Leptospira-

The leptospiral life cycle involves shedding in the urine persistence in the ambient environment

acquisition of a new host and hematogenous dissemination of a new host and hematogenous

dissemination of the kidney though the glomerulus or peritubularcapillaries.

Leptospirosis usually occurs in two Phase;

First phase; (Septicemic phase) about 5-14 days after infection occurs fever, headache, sore

throat, severs muscles, aches in the calves and back and chills occurs suddenly.

Second phase;(Immune Phase)In some people symptoms return a few days later
Classification-

The spirochetal bacteria are divided into two families; Spirochaetes and Leptospireaceae,

respectively. Treponema, Serpulina, and Borrelia are all member of the spirocheateaceae family

the genus Leptospira, which cause leptospirosis, is a member of the leptospirosis family.{17}

Phylum; Spirochaetes

Class; Spirochaetes

Order; Spirochaeles

Species; Leptospira

Family; Leptosiraceae

The genus name Leptospira was first proposed by Noguchi in order to differentiate the Weils

disease spirochete from other known at the time, especially Treponemapallidum, Spirochaete and

Spironema (laterBorrelia) recurrents; the differentiation was based almost entirely on

morphological characteristics. Serogroups and serovars are species and subspeciesof

Leptospirabiflexa, L.kmetys, L.vanthielli, and Wolbachie are among the saprophytic species of

Leptospiraare distinguished bt tests such as pathogenicity to animals.

This new classification will also be sufficient to compare the isolate with reference strain the co-

existence of the two classification is not confusing, thus the species

Leptospirabiflexa,Leptospirainterrogans in the Phenotypic model are also genomospecies in the

genotypic model.
Leptospira species do not multiply outside the host. In the environment, they require high

humidity for survival and are killed by dehydration or temperature greater the 50ºC they are

remain viable for a few to many weeks or months in contaminated soil and for several weeks in

cattle slurry. The precise identification of Leptospira spp. is important for epidemiology and

public health surveillance, as differentserovars can exhibit different host specificities and may

not be associated with a particular clinical form of infection.

GenotypicClassification–

the genotypic classification of Leptospirais supported by multilocus enzyme electrophoresis

data. {18} The multilocus enzyme electrophoresis evidence back up the genotypic species, which

can include both pathogenic and nonpathogenic serovars as well as intermediate species like

L.meyeri, and L.fainei, have been added to the new genomic classification system.{19,20}

Phenotypic classification will be phased out favor of genotypic classification. Indeed, it is based

on a more appropriate taxonomic database than a serological classification. Furthermore, it will

facilitate diagnosis; this new categorization will be sufficient to compare the isolate to strain

references. The coexistence of two classification is not confusing. Thus, the species L. biflexa

and L. interrogans in the phenotypic model are also genomespecies in genotic model.
Pathogenesis-

Leptospira is a blood infection caused by bacteriaLeptospira sign and symptoms can range none

to mild.

Headache

Muscles pain

Fever,

Meningitis or pulmonary bleedings. Weil’s disease is a severe form of leptospirosis that cause

the afflicted person to become jaundiced (Skin and eyes turn yellow). Failure of the kediney

develops. Pulmonary emphysema more than ten genetic type Leptospira produce hemorrhage

syndrome in human, and both wild and domestic animals can spread the disease. The bacteria are

spread to human through animal urine. Coming into contact with the eye, mouth, nose or breaks

in the skin and many symptoms include high fever sever, headache, chills, mussels, aches, and

vomiting, and jaundice abdominal pain, diarrhea if the disease is not treated the patient.

Leptospirosis enters the body through microscopic pores in the skin, and the genital track. For

adhesion and transmembrane passage, chemotaxis mechanism is required. They may however,

settle in the kidneys convoluted tubules and be excreted in urine for a few weeks to month and

occasionally longer.

In susceptible hosts such as humans, system infection can produce severe multi-organ

manifestation. Initial symptoms which may include chills, fever, headache,(severe and

persistence), diarrhea, or a rash myalgia, malaise, prostration, retro- orbital pain, conjunctival

suffusion, muscle tenderness and lung involvement, appear quite abruptly that also have other
symptoms such as meningitis, hemorrhage into skin and mucosa membranes, jaundice,

hepatorenal failure and myocarditis may be misdiagnosed.

The unique feature of Leptospira pathogenesis is the ability of the pathogen to rapidly penetrate

and disseminate during host infection and establish persistence colonization in the in the renal

tubules. It is believed that Leptospira migrate through intracellular junctions. The leptospirosis

spread in rice field workers spend the major of the day in rice fields flooded with water. This

water is often contaminated with rodent urine and Leptospira from urine enters through minor

cuts and abrasions on the hands of these workers making them more prone to get leptospirosis If

the disease is not treated the patient could develop inflammation of the membrane around the

brain and spinal cord, liver failure and respiratory distress in rare cases death occurs. Leptospira

enters the host via small abrasions breaches of the surface integument conjunctival, mucosa

membrane and genital track, is require chemotaxis mechanism for adhesions and transmembrane

passages. However, they may settle in the convoluted tubules of the kidneys and be shed in the

urine for a period of a few weeks to several months and occasionally even longer after the

membrane of leptospirosis in the blood and tissue reaches a critical level, lesions due to the

action of undefined leptospiral toxins or toxic cellular components and consequent symptoms

appear.

The doubling time under optimal conditions is 6-8th and the density is 109 cells\ml culture in

solid medium in slow(except for saprophyte).In susceptible hosts such as humans systemic

infections can produce severe multi- organs manifestations, initial symptoms, which may include

chills, fever, headache ,(sever and persistent), diarrhea or rash, myalgia, malaise, prostration,

retro orbital pain,conjuctival suffusions, mussels tenderness and lung environment, appear quite

abruptly after an incubation period about 10 days. Cases that also have other symptomsand
withouttreatment, leptospirosis can lead to kidney damage, meningitis (inflammation of the

membrane around the membrane around the brain and spinal cord), liver failure, respiratory

disease and even death.

Fig-3 Transmission cycle of Leptopspira

Lab Diagnosis

Leptospiral infection can be diagnosed using blood, urine CSF, infected tissues, and body fluids.

Due to a variety of factors, diagnosing Leptospira is difficult and time-consuming. Due to factors

such as the fact that not all endemic locations have a laboratory. Leptospira’scan be identified in

the blood during the early stages of infection (1-7 days), when a diagnosis is needed to treat the
patient, and then in the urine later on. There is no sensitive, specific, low cost rapid and low cost

widely available diagnostic test for leptospirosis. The diagnosis test for leptospirosis. The

diagnosis is generally made using the MAT on sera. The MAT is difficult to standardize, and is

only performed in three reference and a handful of regional in the world, often lack serovars

infrequently seen in the area. This may result in delayed or missed diagnoses. Since live

organism must be maintained for antigens, it poses a danger to laboratory personnel. Further,

cross- agglutination in common. Diagnosis of Leptospira infection should not be made based on

result of the ELISA test alone, but in conjunction with other clinical sign and symptoms and

other laboratory findings. Epidemiology factors laboratory result should be considered when

making a diagnosis. Leptospira diagnosis have been epidemic area and contact with

contaminated water or animals and blood culture findLeptospira on infection the microorganism

can be found in blood for the first 7- 10 days and then moving to the kidneys.

The first is how to reliably establish the diagnosis. The most common way to diagnose

leptospirosis is through serological tests. Ether the Microscopic Agglutination Test (MAT) and

ELISA (Enzyme Linked ImmunosorbentAssay) which detect serovar specific antibodies on a

solid phase assay for the detection of immunoglobulin IgMantibodies and isolation of Leptospira

from human tissue and body fluid is the creation slandered the leptospirosis. The leptospirosis is

identity the EMJHmedium (Ellinghausen-Mccullough- Johnson-Harris). Bacterial culturing

medium and use stain of silver impregnation technique.

In laboratories that do not see large numbers of cases of leptospirosis or in those without the

capabilities to perform MAT, a commercially availableindirect hemagglutination assay is a

resource that has been demonstrate to have 100% sensitivity and 94% specific for diagnosing

information earlier than MAT. Visualizing leptospiral organism directly in tissue or body fluids
by dark- field microscopy requires a high level of technical expertise and is an inherently low-

yield practice. Culturing the organism is laborious and can take up to two months; nonetheless, at

least two studied have obtained 90% sensitive with blood samples taken early in the course of

infection. It is less common to recover organism from urine probably because Leptospira do not

tolerate acidic environments.

Newer diagnostic includesthe ELISA. One commercial IgM ELISA was found to be 100%

sensitive (93% specific) when compared with the MAT and in 29% of cases detected antibodies

earlier. IgM antibodies were detectable between the 2nd and 6th symptomatic days versus 5th and

8th days for IgG or IgA.

Differential diagnosis-

The divers’ clinical presentations of leptospirosis misdiagnosis. In fact,other illness is initially

suspected in up to 70- 80% of patients eventually found to have leptospirosis. The climatic

conditions other rodent and mosquitoborn infections. In tropical areas, leptospirosis is most

commonly mistaken for dengue fever or dengue hemorrhagic fever. The two infections are

virtually indistinguishable in early stages- fever, headache, and myalgias- and also temporal

distribution due to climatic conditions.

Serological Techniques- To check for antibiotics against Leptospira in the serum of infected

patients, a variety of serological methods are available. This contains the gold

standardMicroscopic Agglutination Test (MAT) An ELISA based commercially accessible

agglutination test. Test kits for detecting IgG and IgM antibodies, as well as a variety of other

products. There are currently, commercially accessible fast diagnostic card test on the market.
Clinical management-

Leptospira causes a non-oliguric hypokalemic form of acute renal insufficiency. Its hallmark

features are impaired proximal sodium reabsorption, increased distal sodium delivery and

potassium wasting. When identified during this initial phase of renal insufficiency, patients have

a better overall prognosis and can be treated with potassium and volume repletion. With

continued sodium and volume loss, however patients develop oliguric renal insufficiency

including acute tubular necrosis. For these patients, dialysis is the critical intervention for

preventing mortality. Timely diagnosis is essential early therapy provides greatest benefit when

initiated early in the course of illness. Although sever late- phase disease can be recognized by

its classic manifestations, identification of early phase leptospirosis is hampered by its non-

specific presentation. Recent findings emphasize that leptospirosis is a frequent cause of

undifferentiated febrile illness in developing countries. Misdiagnosis has become an evenmore

critical issue in regions where dengue and other infectious disease with overlapping presentations

are endemic. Co- infection with disease such as Scrub Typhus and malaria have been reported

and presents another source of confusion in tropical sittings. Identification of leptospirosis will

therefore depend on a high index of suspicion among clinicians and the availability of an

accurate “point of care” test (POC).{21}

Molecular diagnosis-

Molecular diagnostic method is increasingly being used for clinical diagnosis in endemic areas

because of their sensitivity and specificity. PCR amplification techniques should help to

characterize any Leptospira DNA sequence present. Especially in the early stage of an outbreak,

it can be extremely valuable to characterize further any diagnostic DNA sequence that have been
amplified in order to confirm the amplification as being definitively derived from Leptospira and

not due to an anomalous amplification. This can be done by hybridization, restriction

endonuclease digestion or DNA sequencing. Each of addition reagents and equipment and

thusadds to the complexity of the diagnosis process.

Treatment-

For years, the officy of antibiotics in treating leptospirosis has been debated. Leptospira with

their typical bacterial cell walls and ribosomes, are theoretically susceptible to a wide variety of

antibiotics. Tetracycline, was shown to be statistically more effective than placebo introducing

days of fever and symptoms but not in averting renal or hepatic involvement. later penicillin was

found to be significantly more effective than symptomatic management in reducing duration of

fever and symptoms and was most effective when begun within the first 4 days.

Leptospirosis is treated with antibiotics, such as Doxycycline or penicillin, which should be

given early in the course of the disease. Mild leptospirosis is treated with doxycycline,

ampicillin, or amoxicillin. For severe leptospirosis, intravenous penicillin G has long been the

drug of choice, although the third- generation cephalosporins cefotaxime and ceftriaxone have

become widely used. The clinical efficacy of antimicrobials in treating mild as well as severe

Leptospirosis infection is also not well studied and remains a topic of controversy. (22,23,24)

Cephalosporins, Doxy-cycline, and Chloramphenicol have also been used to trat Leptospirosis.

However, for adult patients presenting at the outpatient therapy with mild Leptospirosis using

Doxycycline or Azithromycin an accepted treatment regime. Patient who are allergic to beta-

lactam drugs are treated with amoxicillin or azithromycin in order to avoid use of doxycycline.

Azithromycin and Amoxicillin are also given to children and pregnant women. Severe
Leptospirosis cases requiring hospitalization will be treated with intravenous antibiotics. Though

there are not many clinical trials and reports regarding the development of antibiotic resistance in

Leptospira, this cannot be assumed or presumed that the pathogenic strains remain sensitive to

the prevailing drugs in use.{25,26}

Prevention-

Common prevention measures can include avoiding contacts with rodents, cattle, pigs, and other

domestics animals that are responsible for transmission of Leptospiral infection. Soil agriculture,

lands, and water bodies in endemic regions. Some asymptomatic carrier like rats is resistant to

infections; understandings an appropriate strategy for the control of infection. (27)

Farmers, agricultural workers, freshwater sporting people and people who involved themselves

in flood relief can be guided to use proper protective equipment, like gloves boots etc. to prevent

the contact of the infectious agents. The disease incidence can be reduced or prevented by

avoiding travel to endemic areas where there is a risk of Leptospiral infection. (28)
MATERIAL ANDMATERIAL
Study degien-

This was a prospective study that was conducted in serology section the Department of

Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Science. From March to

November.

Sample collection –

Blood samples of suspected patients were aseptically collected and after separating serum, it was

kept at 4.c

MATERIAL-

ThePanbioLeptospiraIgM ELISA has been demonstrated to detect infections caused by a number

of Leptospirainterrogansserovars including; hardjo, Pomona, Copenhagen.

ManufacturePan Bio diagnose (Germany)

 Materials provided in kit -

Fig-1Matreials provided in kit

Fig-4 PanbioLeptospira kit

 Fig

-5 SAMPLES

Kit content-

* Leptospira antigen coated microwells - ready for use.

* Wash buffer (20x)

* Sample diluent

* HRP conjugate anti human antigen IgM

* TMB chrmogen

* Reactive control

* Calibrator
* Negative control

* Stop solution

Additional material required but not provided -

Accurate adjustable micro pipettors with disposable pipette tips(5 – 1000ul capacity)

1- Deionized water

2- Microplate washing system

3- Microplate reader with 450nm filter

4- Timer

5- Graduated cylinder

6- Flask

7- Test tubes or microtiter plate for serum dilutions

Material required but not provided in kit

1- Incubator

2- ELISA reader

3- Saline water

4- Specimen collection and preparation-

Blood obtained by venipuncture should be allowed to clot at room temperature (20-25) and then

centrifused according to the clinical and laboratorystandards institute (CLSI) (Approved standerd

procedure for collection of diagnositic blood specimen by venipunture H3).

The serum should be separated as soon as possible and refrigerated (2-8c) or stored frozen (<-

20c) if not tested within two days self-defrosting freezers are not recommendedfor storage. The

use icteric sera or sero exhibiting hemolysislipemia or microbial growth is not recommended.

The CLSI provides recommendation for storing blood specimens (Approved standerd- procedure

for handling and processing of blood specimen, H18).

METHOD-

Test procedure-

Ensure all reagents are equilibrated to room temperature (20℃ - 25℃) before commencing

assay. Performing the assay outside the time and temperature ranges provided may produce.

Invalid results assay not falling within the established time and temperature ranges must be

repeated.

ELISA Procedure

Remove the required number of microwells from the foil sachet and insert into strip holder- five

microwells are required for negative control (N), Reactive control (R), Calibrator (CAL) in

triplicate. Ensure the remaining unused microwells are sealed lightly in the foil sachet.
Using suitable test tube or a microtiter plate, dilute the negative control calibrator in triplicate

and patient samples.

To 10μl serum added 1000μl of sample diluent mix well. Alernatively

Pipette 100μl of diluent patient samples controls and calibrator into their respective microwells.

Cover plate and incubate 30 minutes at 30℃

wash six (6) times with diluted wash buffer (Refers to washing procedure).

pippte 100μl TMB into each well.

Incubate for 10 minutes at room temperature (20-25℃) timing from the first addition. A blue

color will develop.

Pipette 100μl of stop solution into all wells in the same sequence and timing at the TMB addition

mix well. The blue color change in yellow.

Within 30 minutes read the absorbance of each well at a wavelength of 450nm with a reference

filter of 600 - 650 nm

Fig-6 ELISA Reader

 Calculation-

Calculate the average absorbance of the triplicate of the calibrator and multiply by the calibrator

factor.

An index value can be calculated by dividing the sample absorbance by the cut-off value.

1- Panbio units can be calculated by multiplying in the index value by 10.

Index value= sampleabsorbance cut-off value

Example; sample A absorbance = 0.949

Sample B absorbance = 0.62

Cut-off value= 0.802× 0.62 = 0.497

Pan Bio units= Index value×10

Sample A 1.91×10 = 19.1 Panbio sample B 0.14×10 = 1.4 Panbio units

INDEX PANBIO UNITS RESULTS

< 0.9 <9 Negative

0.9 - 1.1 9 - 11 Equivocal

1.1 > 11 positive

RESULT INTERETATION

Negative No dectableIgM antibody. If specific IgM antibodies are not

detected and a recent infection is suspected, this can be

confirmed by testing a further specimen 7 - 14 days later.

Equivocal Equivocal samples should be repeated. Samples that remain equivocal

after repeat testing should be repeated by an alternate method or

another sample should be collected.

Positive presence of dectableIgM antibodies

Test Limitation and ex-table values –

The clinical diagnosis must be interpreted with clinical sign and symptoms to the patients. The

result from this kit is not by themselves diagnostic and should be

considered in associations with other clinical data and patients’ symptoms.

Result from immunosuppressed patients should be interpreted with caution.

Screening of the general population should not be performed on patients with clinical symptoms

or when exposure in suspected.

 Population Seroepidemiology may very overtime in different geographical regions.

consequently, the cut-off may require adjustment based on local studies.

The performance characteristics have not been stablished for visual result determination.

The Leptospirosis, IgM antibody is usually present in the first 5-10 days after disease onset. The

presence of specific IgM in a single sample may indicate a recent infection. A positive result in a

single sample should be reported as either suggestive of recent infection or when accompanied

by a supporting clinical presentation a presumptive diagnosis.

Rising level of specific antibody in paired sera can be regarded as serological evidence of recent

infection. The failure to demonstration at least a rise or fall in specific IgMantibodies units in

consecutively collected serum samples, may exclude a recent infection by Leptospiraspecies.

This test is designed as screening test therefore, a small percentage of patients with other acute

infection (e.g. Q fever) may show a positive result. Consequently, all sera demonstrating a

positive result should be referred to reference laboratory for confirmation of IgM specific

antibody and epidemiological recording.

Cross - Reactivity-

A panel of 60 specimens from patients with confirmed disease other than leptospirosis were

tested to established the analytical specificity of the Panbio Leptospira IgM ELISA. The

specimen were from patients with disease that have the potential for cross- reactive. Each of the

specimens include in the study was characterized with respect to disease diagnosis prior to

analysis with the Panbio Leptospira IgM ELISA

 RESULT
Clinical sign, possible risk exposures and laboratory confirmation
This was laboratory – based prospectively study for a period of six- month from March to

November 2021 in Serology laboratory Department of microbiology. A total 170 specimen blood

serum were received the laboratory during the study period.

A total of 170 samples patients clinically suspected for Leptospirosis. Among the 170 samples

available for leptospirosis patients only samples positive by ELISA are confirmed

leptospirosisThe studyinclude170 patient and the results are expressed in both number and

percentage (%). The present study showed the seroprevalence of IgM antibodies out of 170

patients include IPD (In patients Department) OPD (Out patients department) with respect to

different age group.

1- Total patients included in the study = 170.

2- Study period = 15 march to 15 November.

3- Study was conducted in a tertiary care hospital Sanjay Gandhi Post Graduate Institute of

Medical Science (SGPGIMSLucknow).

4- Total number of patients seropositive for Leptospira = 30.

5- Total male 18 (16.3%) and total Female 12 (20%).

6- Total no of Death = 3
7- Risk factor associated with seropositive patients 23% patients of kidney failure, 16%

patients is OP (Out of patients), 40% patients is scrub Typhus positive, and Widal

positive 50% patients is jaundice.

8- The all patients are having fever for more than 15 days.

9- 90% Seropositive patients belong to north India (U.P)

10- Associated with other infections respiratory problem kidney failure, Diabetic problem

and jaundice scrub typhus positive, 2% patients of Dengue positive.

11- Age group associated with maximum Seropositive patients belong to 40 – 60 years 39.4%

patients are seropositive.

80

70 67
64
60

50

40 total sample
age wise total positive
30

20 18
12 13
11
10 8
2 3
0
<10 10-20 yrs 20-40 40-60 >60

Figure-7 Age distribution of Leptospira patients

 Age Group <10 11-20 21-40 41-60 61-100 Total

Leptospira 8 18 64 67 13 170

sample

Percentage% 4.7% 10.58% 37.64% 39.4% 7.6%

Table- 1 Agewisedistribution

Age sex Distribution-

Males contributing to 64% and females being 35% sex ratio----

All cases were in the less then <10 in 7%, 10 – 20 years in 37.64%, 20 – 40 years in 39.4% and

60 – 100 years 7.6% age group.

 Figure-8 Total male and female positive samples

Leptospira Male Female Total

subtypes
Samples 110 60 170

Percentage % 65% 35%

Table- 2

65% male cases and 35% female cases.

Male positive 110 18 16.3%

Female positive 60 12 20%

Total samples positive 170 30 17.64%

 Table-3 Positive and total samples

samples.

110 male
Female
170 total
sample

60

Figure - 9 Total samples of samples

23

OP Patients IP Paitients
 Figure-10 OP (Out patients) IP (In patients)

DISCUSSION-
1-Leptospirosis seroprevalence of almost 28% on infect among human and animal population.

2-Based on our study SeroprevalenceLeptospirosis was total positive 17.64%.

3-Risk factor associated with 23% patients of kidney failure, 5% patients is diabatic problem,

and Liver damagemore than patient respiratory problem and after 15 days illness.

4-Co-infection 2% patient of dengue positive.

Our study shows seroprevalence of leptospirosis around 17.64% while this may be attributed

with is slightly lower compare to other studies better hygiene

Shows mortality of 10% due to leptospirosis patient death.

It is in concordance with other studies.

The burden of leptospirosis in Puerto Ricoremainlargely unknown dispite evidence that the

disease may be underdiagnosed.

The 27.2% seroprevalence observed in among the highest reported in non-occupational related

serosurvege by comparison seroprevalence of 37%was observed among adult males in the

segchelles[29] and 38.2 % and 23.9% of those living in flood prone areas in Bangladesh and

Loae respectively [30,31]

 CONCLUSION-

1- Present history shows increased Seroprevalence of Leptospirosis between August to

October. (Rainy season)

2- ELISA is a rapid easy, and reliable method are not accessible. It is again a species-

specific diagnostic method. Like other disease, PCR is considered a sensitive and specific

method to diagnose Leptospirosis and many techniques are developed from it to produce

more sensitive, specific, and reliable results. Though many techniques are developed,

availability of them in each healthcare concern is questionable in low- and middle-

income countries and resource poor settings. Availability of data over diagnostic results

is also questionable, because many cases remain undiagnosed. Hence, more focus is

needed in these areas where data gap is more.

3- Stringent or continued surveillance and monitoring of Leptospirosis, as emerging agents

is essential to learn more about these divers serotypes domestic animals, living in close

proxitimity to one another in developing countries, for better understanding of

transmission pattern of Leptospirosis that circulate in different geographical locations.

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