CHAPTER – 1 INTRODUCTION

1

.B and Typhoid are some of the serious diseases which are still

public health threat globally. These are major causes of

morbidity and mortality worldwide. Each year millions of new infections

occur which cause more than million death incidences. These diseases are

endemic in many countries of the World. Clinically, it is difficult to

diagnose the disease as, many medical symptoms of both the diseases. For

example, other febrile syndromes such as dengue fever, reckettsial

infection and malaria exhibit many symptoms common to typhoid, besides

that these infections frequently prevail in the same geographic regions.

Mycobacterium tuberculosis complex and nontuberculous mycobacteria

are different clinically. The tuburculi infection symptoms are almost

indistinguishable from pleurisy, pneumonia, chronic bronchitis and other

similar diseases. More than 10 million incidence of tuberculosis are

estimated in 2000 by WHO. Among T.B. patients, 92% suffer from

pulmonary tuberculosis and remaining 8% have other T.B. Many

strategies are adopted to detect these diseases at early stage and with

continuity. Among them serodiagnostic tests are developed with varying

sensitivity and specificity.

An essential component of the disease control program is rapid and

accurate identification of new cases (of the disease). New diagnostic

techniques are urgently needed to confirm the disease with the highest

degree of sensitivity and specificity especially in low income countries

where the diseases (T.B. and Typhoid) are endemic and the incidences are

high. PCR and other amplification technique are too sophisticated and

expensive for routine use in developing countries. We need to develop

T

CHAPTER – 1 INTRODUCTION

2

simple affordable and highly sensitive diagnostic tool, suitable for use in

poor parts of the world.

Antibody response to infectious agent provides useful diagnostic

marker of the infection caused in the host. Specific IgG and IgM antibodies

appear against the specific pathogen. IgM antibodies appear quickly

within some time after the infection but their life / appearance in the blood

remains for short time. IgG antibodies appear later in the primary

response, but in subsequent encounters with the pathogens, these

antibodies are produced quickly and more abundantly which persist for

long time even for years. Serological tests exploit the distinction between

IgM and IgG antibodies.

1.1 THE IMMUNE SYSTEM – THE OVERVIEW

The Immune system is a remarkably adaptive defence system. It

has evolved in vertebrates to protect them from invading pathogenic

microorganisms. The immune system is capable of generating an

enormous variety of cells and molecules capable of specifically recognizing

and eliminating an apparently limitless variety of foreign invaders which

is evident from the majority of infection in normal individual, in them it

appears for a limited duration and leaves very little permanent dosage

(Roitt et al., 1985).

Functionally, an immune response can be divided into :-

1. Recognition

2. Response

Immune recognition is remarkable for its specificity. The immune

system is able to recognize subtle chemical difference that distinguish one

of the foreign pathogens from the other. At the same time, it is capable of

discrimination between “Foreign Molecule” and “Self Molecule” once the

CHAPTER – 1 INTRODUCTION

3

foreign molecule is recognized the immune system enlists the participant

to mount an appropriate response, known as effector response, to

eliminate or neutralize the organism. Thus, the immune system is able to

convert the initial recognition event into different effector responses.

These effector responses are uniquely suited to eliminate a particular

pathogen. Exposer to the same foreign organism at a later stage induces a

memory response.

The immune system can be divided in to two functional divisions.

1. Innate immune system 2. Adaptive immune system

Innate immune system acts as a first line of defence against

infectious agents and potential pathogens and is checked before they

establish an infection.

Adaptive immune system produces a specific reaction to each

infectious agent that normally eradicates that agent. In addition, the

adaptive immune response remembers that particular infectious agent

and prevents from disease or infection at a later stage. (Memory response)

1.1.1 Cells Involve in Immune Response

The cells involve in immune response of vertebrate are of different

type. Which are able to specifically recognize the 'non self‟ antigen on

microorganisms and thereby enhance there elimination. (Table 1.1)

All the cells of the immune system arise from pleouripotent stem

cells with two differentiation (Fig. 1.1)

1. The Lymphoid Lineage – Producing Lymphocytes

2. The Myloid Lineage – Producing phagocytic & other cells

Among different cells involve in immune system, the lymphocytes

and Antigen presenting cells (A.P.Cs) are important for present study,

discussed below.

CHAPTER – 1 INTRODUCTION

4

Table 1.1 : Summary of the cells of immune system

S.

No. Type of Cells

Site of Origin

/Maturation Function

1. Lymphocytes

B Lymphocytes

T Lymphocytes

Null cells

Bone Marrow

Thymus

Humoral Immunity

Cell mediated immunity

Majority of natural killer

cells.

2. Mononuclear

Phagocytic cells

– APC (in the

blood)

Skin, lymphnodes

spleen and thymus

present antigen to

antigen sensitive

lymphoid cell

– Macrophage (in

the tissue)

Alveolar

macrophage

(in lungs)

Phagocytosis

Histiocytes (in the

connective tissue)

Phagocytosis

Kupffer cells (in the

liver)

Phagocytosis

Mesengial cells (in

kidney)

Phagocytosis

Microglial cells (in

the brain)

Phagocytosis

3. Granulocytic cells

Neutrophils Bone marrow Active phagocytic cells.

Eosionophil Bone marrow Active phagocytic cells.

Basophils Bone marrow Non-phagocytic act by

releasing content of their

granules.

4. Mast cells Bone marrow Important role in

development of allergen.

5. Dendritic cells Bone marrow Process and present

antigen to Thymus cells.

Lymphocytes are one among the different type of WBCs produced in

the bone marrow during the process of hemotopoeisis. On leaving from

bone marrow, lymphocytes circulate in the blood and lymph system and

CHAPTER – 1 INTRODUCTION

5

reside in various lymphoid organs. The two major populations of

lympocytes which subserve various function are B-Lymphocytes (B-cells)

and T-Lymphocytes (T- cells).

1.1.2 Organs of Immune System

A number of morphologically and functionally diverse organs and

tissue have various functions in the development of immune response.

They can be divided on the basis of function into:

(a) Primary lymphoid organs

(b) Secondary lymphoid organs

The Thymus and bone marrow constitute the primary lymphoid

organs where maturation of lymphocytes occurs. The lymph node, Spleen

and various Mucosal Associated Lymphoidal Tissue (MALT) constitute the

secondary lymphocytes to interact with antigen. In addition to these,

Tertiary Lymphoid Tissues normally contain free lymphoid cells during

an inflammatory response. Most prominent of these are Cutaneous

Associated Lymphoid Tissue (CALT) and Nasal Associated Lymphoid

Tissue (NALT).

1.2 IMMUNITY

Immunity involves a specific defence response when a forigen

organism or other foreign substance infects host. The immune system

recognizes the foreign substance as not belonging to the body it develops

an immune response against them. Organisms or substances that provoke

such a response are called Antigens. This immune response involves the

production of proteins called Antibodies and specialized lymphocytes.

Immunity can be classified into naturally acquired and artificially

acquired immunity. Artificially acquired immunity accordingly, refers to

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6

the protection that person develops against a certain type of microbe or

foreign substance. Acquired immunity develops daily in individuals

lifetime. Immunity can be acquired either actively or passively. Immunity

is acquired actively when a person is exposed to microorganisms or foreign

substance and the immune system responds.

Immunity is acquired passively when antibody are transferred from

one person to another. Passive immunity lasts only as long as antibodies

are present say from few days to months. Both actively acquired and

passively acquired immunity can be obtained by natural or artificial means.

The various type of acquired immunity are summarized in (Table 1.2).

Table 1.2 : Types of Acquired Immunity

Naturally acquired Artificially acquired

Active Passive Active Passive

Antigen enters

the body

naturally, body

produces

antibodies and

specialized

lymphocytes.

Antibodies pass

through mother

to fetus via

placenta to

infant from her

milk.

Antigens

introduce via

vaccine; body

produces

antibody and

specialized

lymphocytes.

Preformed

antibody by

immune system

and introduced

by injection in

others body.

1.3 HUMORAL AND CELL MEDIATED IMMUNE RESPONSE

1. Humoral response

2. Cell mediated response

The name humoral response was delivered from Latin word

Meaning „body fluid‟ thus humoral immunity can be conferred on a non

CHAPTER – 1 INTRODUCTION

7

immune individual by administration of serum antibody from an immune

individual.

Humoral immunity involves interaction of B cell with antigen and

their subsequent proliferation and differentiation into antibody secreating

plasma cells. Antibody acts as the effector unit of humoral response by

binding to antigen and neutralizing it or facilitating its elimination.

Cell mediated immune response is one in which T cell are

involved. Unlike humoral immunity, cell mediated immunity can be

transferred only by administration of T cell from an immune individual.

Both activated T cell and CTLs serve as effector cells in cell mediated

immune reaction. These effector T cells generates in response to antigen,

are responsible for cell- mediated immunity.

1.4 ANTIGENS

Antigen and antibody play a key role in the response of immune

system. Substances capable of inducing a specific immune response are

commonly refered to as antigens more appropriately immunogen. Most

antigens are either protein or large polysaccharides, lipids and nucleic

acids are antigenic only when combined with proteins and

polysaccharides. Antigenic compounds are often component of invading

microbe such as the capsule, cell wall, flagella, fimbriae and toxins of

bacteria; the coats of virus; or the surfaces of other type of microbes. Most

antigens have a molecular weight of 10,000 or higher. A foreign substance

that has low molecular weight is often not antigenic unless it is attached

to a carrier molecules. These small compounds are called haptens.

Generally, antibodies recognize and interact with specific region in

antigen called antigenic determinant or epitopes.

CHAPTER – 1 INTRODUCTION

8

Immunogenicity and antigenicity are related, but have distinct,

immunological properties that sometimes are confusing. Immunogenicity

is the ability to induce a humoral and or cell mediated immune response.

B cells + Antigen – effector B cells + memory B cells

(plasma cells)

T cells + Antigen – effector T cells + memory T cells

(CTLs)

Antigenicity is the capacity to stimulate the production of

antibodies or cell-mediated immune responces.

1.5 ADJUVANTS

Adjuvants are substance that when mixed with an antigen and are

injected with it, serve to enhance the immunogenicity of the antigen.

Adjuvants are often used to boost when an antigen has low antigenicity.

1.6 ANTIBODIES

Antibodies or protein that are generated in response to an antigen

and can recognize and combine to that antigen. Antibodies can therefore,

help to neutralize or destroy that antigen. Antibodies are highly specific in

recognizing the antigen that stimulate their formation. Antibodies are

members of a group of soluble proteins collectively known as

immunoglobulin (Igs).

1.6.1 Structure of Immunoglubulin

A bivalent antibody has the simplest molecular structure and is

called monomer. A typical immunoglobulin molecule as shown in Figure

has four protein chains; two identical light (L) chains and two identical

heavy (H) chains. The chains are joined by disulphide links and other

bonds to form a Y shaped molecule.

CHAPTER – 1 INTRODUCTION

9

The two sections located at the ends of Y arm are called variable

(V) regions and constant (C) regions. Each antibody has atleast two

identical sites that bind to antigenic determinants. The site are known as

antigen-binding sites. These antigen binding sites are present at the

variable region.

1.6.2 Immunoglobulin Classes

The five major classes of immunoglobulin (Ig) are designated as

IgG, IgM, IgA, IgD and IgE. Each class plays a difficult and distinct role

in immune response.

Structure of Immunoglobulin

1.6.1.1 Immunoglobulin G (IgG)

IgG, the most abundant class in serum, constitutes about of 80% of

the total serum Immunoglobulins. These antibodies are capable of crossing

the walls of blood vessels and enter tissue fluids. There are four IgG

subclasses in humans (IgG1 to IgG4). IgG antibodies offer protection

against circulating bacteria, toxins as well as trigger the complement

system and when bound to antigens, enhance the effectiveness of

phagocytic cells.

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10

1.6.2.2 Immunoglobulin M (IgM)

IgM, account for 5-10% of the total serum immunoglobulin. IgM is

secreted by plasma cells as a pentamer. Its monomeric form is expressed

as membrane bound antibody on B cell. The large size of molecules

prevent IgM from moving about as freely as IgG does. IgM generally

remains in blood vessels and does not enter the surrounding tissue. IgM is

predominant type of antibody involves in response to the ABO blood group

antigens present on the surface of red blood cells. It is also effective in

aggregating antigens. It also can enhance the ingestion of target cell by

phagocytic cells. IgM antibodies are the first antibodies to appear in

response to initial exposer to an antigen and they are relatively short

lived.

1.6.2.3 Immunoglobulin A (IgA)

IgA accounts for only 10-15 % of the total immunoglobulin in serum.

It is the predominant immunoglobulin class in external secretions such as

saliva, tears, breast milk and mucous of the bronchial, gastrourinary and

digestive tracks. In serum, IgA exists primarily as monomer. The most

effective form of IgA is a dimmer called secretary IgA, consisting of two

monomer connected by a J chain. It is produced in this form by plasma

cells in the mucous membrane. Each dimmer then enters and passes

through a mucosal cell, where it acquires a polypeptide called secretary

component and protects it from enzymatic degradation.

1.6.2.4 Immunoglobulin D (IgD)

IgD antibodies constitute about 0.25 of the total serum

immunoglobulin. IgD together with IgM is the major membrane-bound

immunoglobulin expressed by mature B-cells and is thought to function

for the activation of B-cells by antigen.

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11

1.6.2.5 Immunoglobulin E (IgE)

IgE constitutes to about 0.002 % of the total serum antibody. IgE

antibodies mediate the immediate hypersensitivity reactions that are

responsible for the symptoms of hay fever, asthma, hives and anaphylactic

shock. IgE binds to Fc – receptor on the membrane of blood basophils and

tissue mast cells.

1.7 INFECTIVE DISEASES

Infective diseases are caused by microbes that can be passed among

humans by several methods and kill more people worldwide than any

other single cause. Infectious diseases are caused by germs. Germs are

tiny living things that are found everywhere-in air, soil and water. One

can get infected by touching, eating, drinking or breathing, something that

contains a germ. (Specially in case of typhoid and TB)

TB is the third most dangerous infective disease responsible for

human death worldwide. The present study is based on diagnosis of

Typhoid and TB.

1.8 TYPHOID

Typhoid is one of the major infective diseases which is spread

widely in India specially in rural areas. The disease is caused due to a

bacterium which is commonly spread by dirty water and contaminated

food, S.typhi. Large number of cases are reported against S. typhi.

Typhoid fever : A gastrointestinal condition mainly in the developing

world is caused by a bacterial toxin.

Typhoid fever is a life-threatening illness caused by the bacterium

Salmonella typhi.

CHAPTER – 1 INTRODUCTION

12

Life Cycle of Salmonella typhi

CHAPTER – 1 INTRODUCTION

13

Transmission

S typhi has no non human vectors. The following are the modes of

transmission:

Oral transmission via food or beverages handled by an individual

who chronically sheds the bacteria through stool or, less commonly

urine.

Hand-to-mouth transmission after using a contaminated toilet and

neglecting hand hygiene.

Oral transmission via sewage-contaminated water or shellfish.

(especially in the developing world)

An inoculum as small as 100,000 organisms causes infection in

more than 50% of healthy volunteers.

1.8.1 Typhoid and Types

There are two types of Typhoid, paratyphi O and H which are

responsible for having typhoidal infection.

1.8.2 Structure of S. typhi

S. typhi is a gram negative pathogenic bacterium which in human

body creates disturbances and a disease the typhoid. Salmonella can enter

by dirty water or contaminated food.

Early diagnosis of the infection of bacteria is the best, otherwise it

creates a number of problems.

Hardly any receptors are found on Salmonella but on the second

and third layer of cell wall of all strains „O‟ antigen is present which varies

from strain to strain due to change in „O‟ antigenic structure and sugar

composition. The variation in the „O‟ antigen of strains and the antigenicity

of this particular bacteria generate variation in humoral immunity.

CHAPTER – 1 INTRODUCTION

14

Lipid „A‟ present in it is very toxic and is the major cause of fever.

The Vi antigen not only protects the bacteria from being phagocytosed but

enhances virulence of the germ.

1.8.4 Case History of Typhoid In India and other Countries

Typhoid fever is a systemic infection caused by Salmonella enterica

serotype Typhi (S. typhi). The disease remains an important public health

problem in developing countries. In 2000, it was estimated that over 2.16

million episodes of typhoid occurred worldwide, resulting in 2.16 lacs

deaths, and that more than 90% of this morbidity and mortality occurred

in Asia. Although improved water quality and sanitation constitute

ultimate solutions to this problem, vaccination in high-risk areas is a

potential control strategy recommended by WHO for the short-to-

intermediate term.

1.9 SIGNS AND SYMPTOMS

Typhoid fever is characterized by a slowly progressive fever as high

as 40°C (104°F), profuse sweating, gastroenteritis, and non bloody

diarrhea. Less commonly, a rash of flat, rose-colored spots may appear.

Classically, the course of untreated typhoid fever is divided into four

individual stages, each lasting approximately one week. In the first week,

there is a slowly rising temperature with relative bradycardia, malaise,

headache and cough. A bloody nose (epistaxis) is seen in a quarter of cases

and abdominal pain is also possible. There is leukopenia, a decrease in the

number of circulating white blood cells, with eosinopenia and relative

lymphocytosis, a positive diazo reaction and blood cultures are positive for

Salmonella typhi or paratyphi. The classic Widal test is negative in the

first week.

CHAPTER – 1 INTRODUCTION

15

In the second week of the infection, the patient lies prostate with

high fever in plateau age around 40°C (104°F) and bradycardia

(sphygmothermic dissociation), classically with a dicrotic pulse wave.

Delirium is frequent, frequently calm, but sometimes agitated. This

delirium gives typhoid the nickname of "nervous fever". Rose spots appear

on the lower chest and abdomen in around a third of patients. There are

rhonchi in lung bases. The abdomen is distended and painful in the right

lower quadrant where borborygmi can be heard. Diarrheoa can occur in

this stage: six to eight stools in a day, green with a characteristic smell,

comparable to pea soup. However, constipation is also frequent. The

spleen and liver are enlarged (hepatosplenomegaly) and tender, and there

is serum elevation of liver transaminases in blood. The Widal reaction is

strongly positive with anti-O and anti-H antibodies. Blood cultures are

sometimes still positive at this stage. (The major symptom of this fever is

the fever usually rises in the afternoon up to the first and second week.)

In the third week of typhoid fever, a number of complications can

occur:

Intestinal hemorrhage due to bleeding in congested Peyer's patches;

this can be very serious but is usually not fatal.

Intestinal perforation in the distal ileum: this is a very serious

complication and is frequently fatal. It may occur without alarming

symptoms until septicaemia or diffuse peritonitis sets in.

Encephalitis

Metastatic abscesses, cholecystitis, endocarditis and osteitis.

The fever is still very high and oscillates very little over 24 hours.

Dehydration ensues and the patient is delirious (typhoid state). By the end

of third week the fever has started reducing this (defervescence). This

carries on into the fourth and final week.

CHAPTER – 1 INTRODUCTION

16

1.9.1 Type of Diagnostic Test Present

Typhoid (enteric) fever is still a common disease in many developing

countries but current diagnostic tests are inadequate or costly. Studies on

pathogenesis and genomics have provided new insight into the organisms

that cause enteric fever. Better understanding of the microorganisms

explains, in part, why our current typhoid methodologies are limited in

their diagnostic information and why developing new strategies may be a

considerable challenge. The current position of typhoid diagnostics

highlight the need for technological improvements are given below :

Widal Test

Tuberculin Test

1.9.1.1 Tulip Card Test

It is a card test in which antigen of the bacteria is taken in the strip

with which the antibody reacts and gives the positive or negative result,

hence the diagnosis of the disease is done with this normally is

satisfactory but sometimes false results are also reported. Hence by using

the tulip card test the result obtained are not satisfactory. Tulip card test

gives high percentage of false positive results with other microbes.

1.10 TUBERCULOSIS

1.10.1 Introduction

Tuberculosis disease (TB) is a very contagious disease of the

respiratory system. It is a bacterial infection that begins in the lungs and

able to spread by the circulatory system to other parts of the body, such as

the Meninges (the tissue covering the brain), kidneys, and other

abdominal organs and may be fatal. Tuberculosis symptoms are – loss of

appetite and weight, sweating at night, and fever.

CHAPTER – 1 INTRODUCTION

17

Tuberculosis Disease is more often spread (through coughs and

sneezes). In crowded, unsanitary conditions most people who get it have

lowered immunity because of other infections (AIDS) or age. In some

cases, Tuberculosis symptoms may not appear until later in life because

the body immune system has kept the disease under control until the

person becomes elderly and or their immune system is somehow lowered.

Thus this disease is common in some nursing homes where there is crowd

and ventilation is not enough to prevent the spread of TB.

At this point the bacteria enter the respiratory system and it can spread

to other organs if it is not suppressed by the person‟s immune system. A

healthy person is exposed to TB and his immune system is able to contain

the bacteria, it is Latent TB. The latent may become active if the immune

system is somehow impaired, either by age or by other diseases such as AIDS.

The person‟s immune system is able to contain the bacteria in small

sacs or tubercles in the lungs, thus preventing the bacteria from spreading

to other parts of the body where it can cause much damage and even

death. In case of poor or persons with impaired immunity, the bacteria,

through blood may infect organs like kidney, female reproductive organs

and spines. In addition a T.B. sufferer mother is able to spread the disease

to her new born child.

Once TB is spread through the body to other organs, the bacteria

are attacked by the immune system and a minor "war" is started and both

sides loose "warriors." The bacteria die and also healthy tissues die and

turn into growths that are called granulomas. The bacteria are able to live

within these granulomas. However, they do cease growing.

Someone suffering from latent TB is not contagious, asymptomatic

(having no symptoms), feel healthy, however they do have a positive skin

CHAPTER – 1 INTRODUCTION

18

test and the germs of tuberculosis get active without treatment for the

latent TB. In most cases, these persons never get TB, the bacterium

remains inactive through the rest of their life.

1.10.2 History

One third of the world's population is thought to be infected with

Mycobacterium tuberculosis, and new infections occur at a rate of about

one per second. The proportion of people who become sick with

tuberculosis each year is stable or falling worldwide but, because of

population growth, the absolute number of new cases is still increasing. In

2007, there were an estimated 13.7 million chronic active cases, 9.3

million new cases, and 1.8 million deaths, mostly in developing countries.

In addition, more people in the developed world are contracting

tuberculosis because their immune systems are compromised by

immunosuppressive drugs, substance abuse, or AIDS. The distribution of

tuberculosis is not uniform across the globe; about 80% of the population

in many Asian and African countries are positive in tuberculin tests, while

only 5-10% of the US population test positive. The immigrants from

developing countries having latent TB add the incidences of the disease.

1.10.3 The Causes of Tuberculosis

TB is caused by the bacterium Mycobacterium tuberculosis, which is

also called a tubercle bacillus. This bacterium is passed through the fine

spray of water vapor expelled when a person coughs or sneezes.

1.10.3.1 Ptaology of and immunity to tuberculosis

1.10.3.2 Mycobacterium tuberculi

Micobacterium are rod shaped, aerobic, non-spore forming, non

motile bacteria and called acid fast bacilli because they do not stain

CHAPTER – 1 INTRODUCTION

19

readily by gram staining, but once stained they resist decolorization by

acid or alcohol despite being categorized as gram positive bacteria because

they take this particular stain. The mycobacterium genus has a cell wall of

unique composition due to the dominant presence of mycolic acid that

make up more then 50% of its dry weight. The genome of mycobacterium

tuberculosis has been sequenced and shown to be 4.41 Mb in size and to

contain about 4000 protein coding genes of which 52% can be assigned a

function (Cole et al., 1998).

1.10.3.3 Pathology of a immunity to tuberculosis in human

The main route of infection for the tuberculi is the respiratory track.

The bacteria are inhaled in airborn droplet that proceed distally to the

lung to establish an infection (kanufmann, 2001). After entering the lung

the first cell type encountered by the bacteria is the alveolar macrophage,

which is the microbicidal armoury to destroy most potential invaders. The

immune response is initial when Mycobacterium tuberculi arrives in the

alveolar space, where it encounters alveolar microphge. However, the

tubercle bacillus has the extra ordinary ability to persist and even to

replicate in the extreme hostile environment, when most other pathogen

perish, mycobacterium tuberculosis resides in the phagosome, which are

not acidified into lysomes (Clemens, 1996) by inhibitors. Inhibition

secreted by micobacteria and with uptake of micobacteria complement or

mannose binding receptor rather then fc receptor (Schlesinger, 1993). The

inhibition of phagosomal acidifaction occurs by accumulation of a proton –

ATPase (Schaible et al., 1998). Residing in the early recycling endosome,

mycobacterium tuberculosis has ready to access to iron, which is essential

for intracellular survival (Schaible et al., 2002). The pathogenicity of

microbacterium tuberculosis has been attributed to several cell wall

CHAPTER – 1 INTRODUCTION

20

components eg. cord factor a surface glycolipid and lipoarabinomannan

(LAM). Interestingly, the macrophage mannose receptor (MMR) binds to

the virulent mycobacterium tuberculosis strain H37RV but not the

avirulant strain H37Ra, although both strains contain the same amount

of terminal dimannosyl residues ( Schlesinger et al., 1994, Schlesinger et

al., 1996). A major hemopolysacchride, LAM inhibits macrophage

activation by TNF alfa and includes macrophage to secret tumor necrosis

factor alfa (TNF alfa) and interlukin 10 (IL-10) (Barnes et al., 1992).

Lectin DC specific intracellular adhesion molecule-3 grabbing non intgrin

(DC-SING) is also known as a micobacterium tuberculosis receptor on

human DCs (Tailleux et al., 2003). The tuberculi bacillus and its cell wall

glycolipid lipoarabinomannan seem to bind to and to induce via-DC-

SING, an intracellular signal leading to IL-10 production which inturn

impairs activation of protective T cell response derived against

mycobacterium tuberculosis. Ingestion of mycobacterium tuberculosis by

macrophage is also believed.

Ingestion in lysosome does also depend on the engagement of TLRs,

TLRs2 and TLR4. However, the role of TLRs is still controvertial, since

there are contradictory results from TLR-2 or TLR-4 deficient mice i.e.

survival of microbacteria in the suicidal bag (Abel et al., 2002; Reiling et

al., 2002; Shim et al., 2003).

The bacteria enter the parenchyma and can replicate within the

alveolar macrophage or in resident lung macrophage. The signal induced

result in migration of monocytes drived microphage and resident DCs to

the focal site of infection in the lungs. Immunohistochemical i.e

microscope, histochemical, flowcytometer analysis show that Micobaterium

bovise BCG purified protein derivative (PPD) beeds mobilized CD11c +

CHAPTER – 1 INTRODUCTION

21

DCs of comparable maturation transfer of DCs from PPD antigen

challenged lungs conferred a Th1 anamnestic cytokine response in

recipient (Chiu et al., 2004) Once the, CD4+ and CD8+ T cells are primed

against micobacterial antigens primed T cells expand and migrate back to

the lung to the focus of infection, presumably in response to signal such as

chemokines produced by or in response to infected cell (Gonzalez and

Orme, 2001).

Formation of compact granulomas that contain the pathogens at

these sites begin with the accumulation of macrophage at sites of bacterial

implantation and multiplication (Dannenberg, 1989). The migration of

macrophage and T cells, as well as B cells, to the site of infection

culminates in the formation of a granuloma, a characteristic feature of

tuberculosis. In addition of T cells and macrophage the granuloma consists

of other host cells including B cells, dendritic cells, endothelial cells and

fibroblast (Gonzaler and Orme, 2001). The granuloma can show central

caseous necrosis and give rise to cavities although this dose not occur in

all cases of TB patients. As a key aspect of granuloma formation the

development of fibrosis within the granuloma and in surrounding

parenchyma, which produces microscopic nodules (tubercles) occurs. The

massive activation of macrophage that occurs within tubercle after results

in the concentrated release of lytic enzyme (Chandrasekhar and

Mukherjee, 1990; Converse et al., 1996). These enzymes destroy nearby

healthy cells, resulting in circular region of necrotic tissue, which

eventually form a lesion with caseous consistency. As these caseous lesions

heal, they become calcified and are radially visible on X ray, where they

are called Ghon complexes. In adults, the disease advance as necrotizing

pneumonis process that can involve and result in spread of infection to

others area of lungs (North and Jung, 2004).

CHAPTER – 1 INTRODUCTION

22

Tuberculosis immunity relies mainly on cell mediated immunity

rather than humoral immunity. The acquired cellular immune response

to mycobactium tubercuosis is complex CD4+ and CD8+ T cells as well as

inconventional T cells such as γ T cells and CD restricted CD4 – CD8 or

CD4 / CD8 single positive antibody T – cell subgenerally sets and NK

cells are involved (Suzuki et al., 1986;, Schaible et al., 2000;, Shen et al.,

2002), but generally CD4 T cells play a central role in protection.

Interferon - γ is a key cytokine in the immune response against

mycobacterium tuberculosis (Flynn et al., 1993). This is demonstrated by

the considerably increased risk of tuberculosis patient with reduced cell

mediated immunity, such as these infected with HIV or individual

undergoing immunosuppressive therapy. Here, by comparing patients

with defective humoral immunity, such as these with multiple myeloma,

which show no increased predisposition to Tuberculosis (Cohen et al.,

1987). The patients who were deficient of IL 12 Rc signaling and INF–g

production suffered from severe micobacterial and salmonella infection

(D Jong et al., 1998).

The macrophage has multiple function in Tubercuosis including

antigen processing and presentation and effector cell function. Ingestion of

Micobacterium tuberculosis by macrophage triggers, via NF-kB activation,

transcription of numerous macrophage gene including these that code for

proinflammatory cytokine and chemokine. The infected macrophage

releases TL-12 and IL-18, which stimulate T lymphocytes predominantly

CD4 + T cells to releases IFN-γ (Wang et al., 1999). However, atleast in

the mouse model of infection Micobacterium tuberculosis has the ability to

evoke the inslaught of innate immunity, as virulent bacilli replicate

exponentially.

CHAPTER – 1 INTRODUCTION

23

1.10.4 Infection and Transmission

Tuberculosis (TB) is a contagious disease. Like the common cold it

spreads through air. Only people who are sick with TB in their lungs are

infectious. When infectious people cough, sneeze, talk or spit, they propel

TB germs, known as bacilli, into air. A person needs only to inhale a small

number of these to be infected.

Left untreated, each person with active TB disease will infect on an

average between10 and 15 people every year. But people infected with TB

bacilli will not necessarily become sick with this disease. The immune

system “walls off” the TB bacilli which are protected by a thick waxy coat,

can lie dormant for years. When someone‟s immune system is weakened,

the chances of becoming sick are greater.

Some in the World is newly infected with TB bacilli every second.

Overall, one-third of worlds population is currently infected with TB

bacillus.

5-10% of people who are infected with TB bacilli (who are not

infected with HIV) become sick or infected at some time during there

life. People with HIV and TB are much more likely to develop TB.

1.10.5 Global and Regional Incidence

WHO estimates that the largest number new TB cases in 2008

occurred in the South East-Asia Region, which account for 34% of incident

cases globally. However, the estimated incident rate with sub-Saharan

Africa is nearly twice that of the South- East Asia Region, with over 350

cases per 100000 population. An estimated 1.3 million people died from TB

in 2008. The highest number of deaths was in South East-Asia Region,

while the highest mortality per capita was in the Africa Region.

CHAPTER – 1 INTRODUCTION

24

In 2008, the estimated per capita TB incidents was stable or falling

in all six WHO regions (Table 1.3). However, the slow decline in incidence

rates per capita is offset by population growth. Consequently, the number

of new cases arising each year is still increasing globally in the WHO

regions of Africa, the Eastern Mediterranean and South East-Asia.

Table 1.3 : Estimate TB incidence, prevalence and mortality, 2008

(WHO fact sheet)

WHO

region

Incidence1 Prevalence2 Mortality

No. in

thou-

sands

% of

global

total

Rate

per

100000

pop3

No. in

thou-

sands

Rate

per

100000

pop3

No. in

thou-

sands

Rate

per

100000

pop3

Africa 2828 30% 352 3809 473 358 48

The

America 282 3% 31 221 24 29 3

East

Mediterr

anean

675 7% 115 929 159 115 20

Europe 425 5% 48 322 36 55 6

South-

East Asia 3213 34% 183 3805 216 477 27

Western

Pacific 1946 21% 109 2007 112 261 15

Global

Total 9369 100% 139 11093 164 1322 20

1 Incidence is the number of new cases arising during a defined period.

2 Prevalence is the number of cases (new and previously occurring) that

exists at a given point in time.

3 Pop indicates population.

CHAPTER – 1 INTRODUCTION

25

1.10.6 HIV and TB

HIV and TB (together) form a lethal combination, each speeding the

other‟s progress. HIV weakens the immune system. Someone who is HIV-

positive and infected with TB bacilli is many times more likely to become

sick with TB then someone infected with TB bacilli and HIV-negative. TB

is a leading cause of death among people who are HIV-positive. In Africa,

HIV is the single most important factor contributing to increase the

incidences of TB since 1990. WHO and its international partners have

formed the TB/HIV Working Group to develop global policy on the control

of HIV-related TB and to advise and tackle as how to fight against TB and

HIV activities and the lethal combination. The interim policy on

collaborative activities describes steps to create mechanisms of

collaboration between TB and HIV/AIDS programmes, to reduce the

burden of TB among people and reducing the burden of HIV among TB

patients.

1.10.7 Drug- resistant TB

Global tuberculosis control is facing major challenges today. In

general, much effort is still required to make quality care accessible

without barriers of gender, age, type of disease, social setting and ability

to pay. Co-infection with Mycobacterium tuberculosis and HIV (TB/HIV),

especially in Africa, and multidrug-resistant (MDR) and extensively drug-

resistant (XDR) tuberculosis in all regions, make control activities more

complex and demanding. Several risk groups need special attention and

technical approaches.

Until 50 years ago, there were no medicines to cure TB. Now,

strains that are resistant to a single drug have been documented in every

CHAPTER – 1 INTRODUCTION

26

country surveyed; what is more, strains of TB resistant to all major anti-

TB drugs have emerged. Drug-resistant TB is caused by inconsistent or

partial treatment, when patients do not take all their medicines regularly

for the required period because they feel better, because doctors and

health workers prescribe the wrong treatment regimens, or because the

drug supply is unreliable. A particularly dangerous form of drug- resistant

TB is multidrug- resistant TB (MDR-TB), which is defined as the disease

caused by TB bacilli resistant to at least isoniazid and rifampicin, the two

most powerful anti-TB drugs. Rates of MDR-TB are high in some

countries, especially in the former Soviet Union, and threatening TB

control efforts.

While drug resistant TB is generally treatable, it requires extensive

chemotherapy (up to two years of treatment) when the second line anti-TB

drugs which are more closely than first line drugs, and which produce

adverse drug reactions that are more severe, though manageable. Quality

assured second-line anti-TB drugs are available at reduced prices for

projects approved by the Green Light Committee.

The emergence of extensively drug-resistant (XDR) TB, particularly

in settings where many TB patient are also infected with HIV, pose a

serious treat to TB control, and confirms the urgent need to strengthen

basic TB control and to apply the new WHO guidelines for the

programmatic reduction of drug- resistant TB.

1.10.8 Signs and Symptoms of Tuberculosis

The respiratory system is the most common place where the

bacteria grow. Respiratory symptoms may include horrible coughing that

extends longer than three weeks, chest pain, blood in sputum when

CHAPTER – 1 INTRODUCTION

27

coughing. Other symptoms include extreme fatigue, reduced body weight,

loss of appetite, high fever, chills, night sweats.

In addition, TB results in abnormal chest x-rays and lab results. It

must be remembered that a person with active TB can spread it easily to

others nearby.

Mycobacterium tuberculosis. Acid-fast stain (1000X)

1.10.9 Type of Diagnostic Test Present

Sputum used to detect TB. The most widely used technique for

diagnosing active tuberculosis is sputum smear microscopy: a person

suspected to have TB produces a sputum sample, coughed up from their

lungs, that is examined under a microscope, for evidence of TB

mycobacteria. Rapid simple, accurate and attendable diagnosis of TB in

new cases is most essential to control Tuberculosis. Hence, cheap, reliable

and fast diagnostic techniques are frequently needed which may fecilitate

microscopy in countries like India, Bangladesh, Nepal etc.

ELISA, RIA, FCR are some reliable and fast detecting tools but are

quite expensive for routine examination in developing countries. Here the

CHAPTER – 1 INTRODUCTION

28

merits of some techniques are discussed. The diagnostic test for TB are

divided in to direct and indirect methods.

Microscopic detection, culture, immuno assay for detection of

antigen. FCR of bacterial DNA or RNA (rRNA), tuberculostearic acid and

other tubercle lipids detection by gaschromatography – mass spectrometry

are the direct method.

Skin test (tuberculin test) stimulation assay of lymphocytes and

measurement of antimicrobacterial antibodies come under indirect method.

Microscopy: Sputum Microscopy Ziehl: (1879) developed a simple

staining technique which was later modified by Nelsen in 1883. This is

considered to be a rapid and most widely used in sputum of a person

suspected to have TB infection. A positive ZN indicates the presence of

mycobacterium.

Microscopy can be done on tissue biopsies, urine, sputum and

cerebrospinal fluid (CSF) but it is less sensitive. In the sputum of

respiratory tuberculosis person, the test is highly specific but the

sensitivity rate varies.

Culture : It is also known as “Gold Standard” method for TB diagnosis.

Even in smear positive sputum growth of bacteria takes 3 to 8 weeks.

Some improvements have been made using a liquid media and indicator

etc. Decontaminants and antibiotics are in use these days.

Monoclonal or polyclonal antibodies is a cheap method for

identification of TB antigen. Besides, X-ray and abreugraphy, tuberculin

test nucleic acid amplification etc, are some of the other tests.

Nucleic acid amplification: PCR is based on amplification of TB.

DNA or RNA in clinical samples. IS 6110 is specific for strains belonging

to the M.tuberculosis complex.

CHAPTER – 1 INTRODUCTION

29

This method is highly specific and more sensitive. Results can be

obtained within 24 hrs. It is valuable for the diagnosis of other types of

TB. PCR test kits cannot be used for routine laboratory test in third world

nations as it requires good laboratory facilities, well trained staff, regular

supply of very costly probes etc.

Detection of tuberculostearic acid, mycobacterial antigens

Glycolipids, lippopolysaccharides defined proteins antigens of M.

tuberculosis are other methods of identification with their limitations,

specificity and sensitivity.

Indirect Methods : Measurements of the humoral response is done by

detecting specific anti-TB antibodies. A good amount of work has been

done with antigens 5, 6, A60, DAT (diaacyltrehalo) 85A and 38 Kd

antigen. For increased sensivity, and their detection, ELSA and Western

blotting are essential requirement.

ELISA based assays have high sensitivity only in smear positive

cases.

Antibody assay are relatively simple, cheap and many samples can

be examined at a time.

Diagnosis of TB by Microscope : Testing for TB with a microscope

can at best only reveal whether the TB mycobacteria are present or not in

the sputum of a patient. In the case of people with drug-resistant TB,

further tests are necessary to pinpoint the particular resistant strain

infecting the patient. This can only be carried out at present by growing

the bacilli in a well-equipped laboratory. The bacilli then have to be tested

to find out which drugs they are resistant to in laboratory conditions. Drug

resistance can be more rapidly detected by new molecular tests but these

again require excellent laboratory conditions. These kinds of diagnostic

CHAPTER – 1 INTRODUCTION

30

tests just can not be done in the remote areas where MSF works. The

length of time it takes to get the results means many patients do not get

the correct treatment in time. In the case of XDR-TB, most patients will

die before they can be correctly diagnosed. Diagnosing people who are HIV

positive is also extremely difficult because there are less bacteria in their

sputum and the disease is more prevalent outside the lungs in these cases.

The result is that many patients going undiagnosed and die.

Radiography :

Chest X-ray showing TB

Tuberculosis creates cavities visible in x-rays like this one in the

patient's right upper lobe.

In active pulmonary TB, infiltrates or consolidations and/or cavities

are often seen in the upper lungs with or without mediastinal or hilar

lymphadenopathy or pleural effusions (tuberculous pleurisy). However,

lesions may appear anywhere in the lungs. In disseminated TB a pattern

of many tiny nodules throughout the lung fields is common - the so called

miliary TB. In HIV and in other immunosuppressed persons, any abnormality

may indicate TB or the chest X-ray may even appear entirely normal.

Abnormalities on chest radiographs may be suggestive of, but are

never diagnostic of TB. However, chest radiographs may be used to rule

out the possibility of pulmonary TB in a person who has a positive reaction

to the tuberculin skin test and no symptoms of disease.

CHAPTER – 1 INTRODUCTION

31

Cavitation or consolidation of the apexes of the upper lobes of the

lung may be discernible by a chest x-ray.

Abreugraphy : A variant of the chest X-Ray, abreugraphy (after the

name of its inventor, Dr. Manuel Dias de Abreu) was a small radiographic

image, also called miniature mass radiography (MMR) or miniature chest

radiograph. Though its resolution is limited it is sufficiently accurate for

diagnosis of tuberculosis.

Much less expensive than traditional X-Ray, MMR was quickly

adopted and extensively utilized in some countries, in the 1950s. For

example, in Brazil and in Japan, tuberculosis prevention laws enforced

into effect, obligating ca. 60% of the population to undergo MMR

screening.

The procedure went out of favor, as the incidence of tuberculosis dramatically

decreased, but is still used in certain situations, such as the screening of prisoners

and immigration applicants.

Tuberculin skin test (TST) : Two tests are available : the Mantoux and

Heaf tests.

Mantoux skin test:

Injecting a Mantoux skin test

CHAPTER – 1 INTRODUCTION

32

Mantoux test

The Mantoux test for TB involves intradermally injecting PPD

(Purified Protein Derivative) tuberculin and measuring the size of

induration 48-72 hours later.

The Mantoux skin test is used in the United States and is endorsed

by the American Thoracic Society and Centers for Disease Control and

Prevention (CDC). If a person has had a history of a positive tuberculin

skin test, another skin test is not needed.

Heaf test:

The Heaf test was used in the United Kingdom until 2005, and is

graded on a four point scale. The Mantoux test is now used.

The equivalent Mantoux test positive levels done with 10 TU (0.1

ml 100 TU/ml, 1:1000) are

0–4 mm in duration (Heaf 0 to 1)

5–14 mm in duration (Heaf 2)

Greater than 15 mm in duration (Heaf 3 to 5)

CDC classification of tuberculin reaction : An in duration

(palpable raised hardened area of skin) of more than 5–15 mm (depending

upon the person's risk factors) to 10 Mantoux units is considered a positive

result, indicating TB infection.

5 mm or more is positive in –

CHAPTER – 1 INTRODUCTION

33

o HIV-positive person

o Recent contacts of TB case

o Persons with nodular or fibrotic changes on CXR consistent

with old healed TB

o Patients with organ transplants and other immuno

suppressed patients

10 mm or more is positive in –

o Recent arrivals (less than 5 years) from high-prevalent countries

o Injection drug users

o Residents and employees of high-risk congregate settings

(e.g., prisons, nursing homes, hospitals, homeless shelters, etc.)

o Mycobacteriology lab personnel

o Persons with clinical conditions that place them at high risk

(e.g., diabetes, prolonged corticosteroid therapy, leukemia,

end-stage renal disease, chronic malabsorption syndromes,

low body weight, etc)

o Children less than 4 years of age, or children and adolescents

exposed to adults in high-risk categories

15 mm or more is positive in –

o Persons with no known risk factors for TB

o Targeted skin testing programs are conducted among high-

risk groups

A tuberculin test conversion is defined as an increase of 10 mm or

more within a 2-year period, regardless of age.

1.10.10 Problems Related to Test

CHAPTER – 1 INTRODUCTION

34

Skin test may create suspicion between active T.B and infection of

M.tuberculosis without disease. Geographical areas, status of BCG

vaccination, previous exposure to the pathogen, malnutrition, immune-

suppression, age and co-existence of disease are some of the factors affect

the responsiveness of Skin test.

Some more specific skin test are aimed to develop. MPB64

considered to be a more potent skin test. It gives positive result in 52 of 53

active TB patients. ESAT-6 another antigen from the bacillus is a

potential diagnostic reagent (200,201). Culture protein filtrate (CPF) 10,

gamma interferon assay, LAM and PGL-1 assays are more indirect

methods. Their sensitivity and specificity in T.B. cases require exhaustive

survey. Skin test is simple and cheap but cannot be reliable where people

are vaccinated with BCG.

The Aim of Work –

Present study is to develop an easy, cheap, faster and reliable kit

with high sensitivity and specificity to T.B. and also to deduct the

Mycobacterium tuberculosis in healthy person in whom the symptoms of

the disease have not appeared. Liposome coated with antigen from T.B.

and their reaction with specific antibodies which finally give a visual

colouring reaction is one of the aims of the diagnosis. Such assay would

make a significant contribution to early diagnosis, treatment of diseases

like typhoid and T.B. on a simple and inexpensive format.

The similar strategy is adopted for developing the diagnostic tool for

typhoid. Different types of antigens of S.typhi are tagged with the

liposome containing chromagen with the idea that some of them would

react to some particular antibody to release the colour.

CHAPTER – 1 INTRODUCTION

35

1.11 LIPOSOMES

The Liposomes are discovered by sir Bangham in the year 1965 and

basically developed for drug delivery. It is firstly used to study in-vivo

simulated biomembrane behavior and excessively used as tool for drug

delivery and targeting. It also covers areas like bioreactor, catalyst,

cosmetics, ecology and also used in some tumor targeting gene and anti-

sense therapy.

Liposome are concentric bilayer vesicles in which aqueous volume is

entirely enclosed by membranous lipid bilayer composed of natural or

synthetic phospholipids.

1.11.1 About Liposomes

Liposomes are basically developed for drug delivery system. They

were first used to study in vivo for simulated boimembrane behaviour and

now vigorously used as a tool for drug delivery and targeting. It also

covers area like bioreactor, catalysts, cosmetics and ecology. Now a days, it

is also used for drugs in tumor targeting, gene and antisense therapy.

Basically liposome are concentric bilayer vesicles in which aqueous volume

is entirely enclosed by membranous lipid bilayer composed of natural or

synthetic phospholipid.

1.11.2 Mechanism of Formation of Liposome

Liposome are formed when phospholipids are hydrated

phospholipids are amphipathic in nature (i.e they are having

affinity for both aqueous and polar solvents)

it has hydrophobic tail and hydrophilic or polar head

CHAPTER – 1 INTRODUCTION

36

the tail of liposome consists of 10 to 24 carbon and 1 to 6 bonds in

each chain

its polar or phallic end is mainly phosphoric acid bound to water

soluble molecule.

in aqueous medium the liposome itself arranges so that the polar

end of it comes in contact with the polar part of environment and

shields non polar part

at high concentration of these polar lipids, liquid crystalline phase

is formed that up to dilution.

1.12 TYPHOID

In many countries and also in many persons the diagnosis of

typhoid remains a problem because of the sensitivity, false positive and

time limitation of Widal test. Besides, the clinical symptoms create

confusion with that of Malaria, dengue, and other febrile illnesses hence a

need to develop new serological diagnostics was greatly demanded House-

et al., 2001, studied many receptors and detection of antibodies against

them in populations. I CDDR (2003) studies proteins lipoproteins and

polysaccharides of the bacteria and detection of antibodies against them.

Olsen et al., 2004 developed a rapid diagnostic test for typhoid fever based

on IgM antibodies and reaction of indicators particles by magnetic

separation. The test was based on the detection of antibodies against

lipopolysaccharide which was originally developed by Lim et al., (1998)

interestingly, the test detected only IgM but not IgG (Tam and Lim, 2003)

their new method for detection of typhoid was based on particle inhibition

immuno assay. This test has its limitation in the sense it dose not detect

anti 09 antibody and IgG. Tam and Lim 2003 developed a detection

method based on particle separation in tubes this is a 2 min test but had

CHAPTER – 1 INTRODUCTION

37

its limitation for the development of antibodies and clinical symptoms of

the patients. Wain et al., 1998 quantified bacteria in blood of patients and

found out relation ship between bacterial count and clinical features. The

number of bacteria detected in the blood of patient was not in conformity

with the clinical features of the disease, it was positive in 40 to 50% cases.

Carlsson et al., 1975 applied ELISA in quantifying half „O‟ antibodies

against salmonella in human serum. 09 epitope is immuno dominant

which could be detected by this method this antibody is directed against

the LPS epitope later on in 2007.

Pastoor et al., 2007, developed a simple rapid test for Typhoid based on

serodiagnosis confirmed by culture test but highly varied sensitivities

were obtained which also depended on duration of illness yet less specific

then Widal Test.

Frankie Tam et al., 2008, developed 5-min colorimetric test for typhoid

and para typhoid which actually detects 09 antibody with good sensitivity

in both case and made a conclusion that diagnostic accuracy increases if

both TUBEX –PA and TUBEX-TF are applied.

Dutta et al., 2006, tried to develop a typhoid test, the tubex in which

lipopolysacchride antibody (anti To) and anti-Typhi H flagella antibody

(anti TH) titer were taken. They also calculated a time, sensitivity

Positive predictive value (PPV) Negative Predicted value (NPV) Its results

were good i.e true positive in typhoid and true negative were obtained in

malaria. 69% PPV and 77% NPV were observed.But they concluded that

sensitivity, specificity, PPV and NPV of Typhoid and Tubex were not

better then Widal test. Nakhla et al., 2011 moved to trap typhoid by two

diagnostic field test a latex agglutination Dri- Dot assay and IgM Lateral

flow assay. Both tests were done separately gave reduced sensitivity and

CHAPTER – 1 INTRODUCTION

38

specificity but the limitation of both the tests was their sensitivity at early

stage of disease. Both the tests when performed parallel, they increased

sensitivity up to 85% but decreased specificity up to 70% was noted. The

biggest drawback with this is, patient with longer duration of illness did

not give good results for diagnosis. Pastoor, 2007 performed rapid latex

agglutination assay for serodiagnosis of typhoid which was basically good

in trapping the typhoid but was not showing good specificity.

Rahman et al., 2007, developed a 5 minutes detection i.e 5 min TUBEX- R

09 antibody detection kit which was mostly experimented on children and

infants. TUBEX- R was 82.9%, (172/209) specific and 91.2 % was (31/34)

sensitive in febrile subjects. Specificity was found better in non- febrile

(89.5 %) and suggested that culture negative febrile individual could truly

be typhoidal. These were also positive with anti- crude 09 ELISA and

Widal test. But drawback of the test was, it did not detect both IgG and

IgM antibody.

Kasper et al., 2010 conducted an etiological study among combodian

patients. Salmonella enterica serovar typhi was detected in 0.9% blood

cultures and assumed to be responsible for fever. Antimicrobial

susceptibility testing showed decrease susceptibility for ampicillin (56%),

choramphenicol (56%), nalidixic acid (81%), ciprofloxacin (0%) and reduced

susceptibility up to 80 % was obtained, with the technique.

House et al., 2001 did the serological test regarding the Typhoid fever in

an area of endemicity and obtained very good sensitivity with the test in

the patients. This test was totally based on the serology so obviously had

some of the limitations. Wain et al., 1998, made bacterial quantification in

blood of Typhoid fever patients.

CHAPTER – 1 INTRODUCTION

39

1.13 TB

Microscopic culture, nucleic acid amplification, gas liquid chromatography

– mass spectrophotometry are various methods for detecting Tuberculosis.

These methods have been developed and cross checked by many scientists.

In 1882 Tuberculi bacillus was first isolated by Robert Koch. Ziehl-Neelsen

developed simple staining test for detection of microbacterial infection but

it was not the diagnostic tool of TB. Microscopic detection of TB in sputum

is highly specific but sensitivity rate vary between 34% to 79% (Lipsky et

al., 1984; Gordin and Slutkin 1990; Elliott et al., 1993; Karstaed et al.,

1998; Levy et al., 1998). In non respiratory TB, microscopy was not of

much use. Lipsky et al., 1984; Gordin and Slutkin 1990; Nguyen et al.,

1999 reported many false negative results which could only be reduced

with the help of computerized directed automated microscopy, (Somoskovi

et al., 1999) this is a costly instrument. Centrifugation of sputum was used

by Saceanu et al., 1993; Miorner, 1994; Habeenzu et al., 1998; Warren et

al., 2000 to increase the sensitivity of the test. Flotation technique to

concentrate mycobacterium in sputum by organic solution was applied by

Kinyoun 1915; Rattan et al., 1994. There are ample chances with sputum

of patients who are confirmed with HIV with the help of microscope to get

micobacteria but species cannot be identified (Gordin and Slutkin 1990;

Long et al.,1991; Githui et al ., 1992; Elliott et al., 1993).

Culture is also known as gold standard method and widely used for

diagnosis of T.B. The disadvantage with this technique is slow growth of

microbacteria. Many modifications have been done by using different

chemicals to increase the growth of mycobacterium only (Kubica et al.,

1963; Middlebrook et al., 1977; Bird et al., 1996; Mejia et al., 1999).

CHAPTER – 1 INTRODUCTION

40

BACTEC 460 system to automated MCIT method have been developed for

visual reading of the presence of bacteria, these methods are also based on

detecting radioactive carbondioxide but again the cost of the instrument,

its handling and highly costly chemicals are the drawbacks.

Isolations and identification of micobacteria is very slow process and need

many more days for growth and identification Species specific DNA probes

such as nonradiometric accuprobe (USA) has surprisingly reduced time

and also the identification of various species of mycobacterium can be done

from colonies (Gonzaler and Hanna, 1987; Musial et al., 1988). Molecular

amplification methods such as Polymerase Chain Reaction are developed

by Wobeser et al., 1996; Ninet et al., 1999 to identify mycobacterium from

its culture.

Mycolic acid is a metabolite synthesized by mycobacterium species the

mycolic acid is species specific. The identification of the acid can be done

through High Performance Liquid Chromatography(HPLC) or thin layer

chromatography(TLC) these methods are highly accurate but are used

mainly in reference and research laboriteries (Butler et al., 1991; Liquin et

al., 1991; Glickman et al., 1994). These require considerable expertise and

expensive equipments.

A rapid identification of mycobacterium by a cheap method can be done by

micobacterial antigen in cultures by polyclonal and monoclonal antibodies

(Friedman et al., 1989; Schoningh et al., 1990; Verstijnen et al., 1991;

Krest‟anpol et al., 1993; Liashchenko et al., 1997). The maximum clinical

samples do contain Micobacteruim tuberculosis which can be rapidly

screened by MOTT test. In 1992 center for disease control and prevention

recommended BACTET/ NAP/ HPLC methods (Hinman et al., 1992).

CHAPTER – 1 INTRODUCTION

41

Nucleic acid amplification, by polymerase chain reaction is based on the

detection of amplified DNA or RNA in clinical samples. Oligonucleotide

primers are used to direct the amplification of target nucleic acid sequence

by repeated rounds of denaturation, primer annealing and primer

extension (Millis and Faloona, 1987). Most commonly used target is IS

6110 (Thierry et al., 1990; Eisenach et al., 1990; Brisson-Noel et al., 1991;

Kolk et al., 1992; Kox et al., 1994). This sequence is specific for strains

belonging to the M.TB complex and up to 20 copies are present in

microbial genome. Its advantage is to give results in 24 hours. The

technique is highly sensitive and specific. Polymerase chain reaction of

cerebrospinal fluid is the best method for laboratory diagnosis of TB

meningitis (Shankar et al., 1991; Kox et al., 1995; Nguyen et al., 1996).

The diagnosis of tuberculostearic acid in clinical samples using gas

chromatography – mass spectrophotometery with selective ion monitoring

is good tuberculosis detection technique. Good results are reported by

detecting TBSA in cerebro spinal fluid and sputum (French et al., 1987)

though results with pleural fluid were poor (Yew et al., 1991). The

drawback with these methods is complexity of technique and expensive

equipments.

Sada et al., 1983, described the detection of microbial antigen in cerebro-

spinal fluid of patients with tuberculous meningitis by ELISA. The

specificity of the test was 90 % in meningitis of other causes and 100 % in

control subjects. Shaoli Deng et al., 2011, evaluated the diagnostic test

detecting antibodies against lipoarabinomannan, 38 KDa antigen of

mycobacterium tuberculi. By using these protein chips 160 tuberculosis

patients and 150 normal subjects were detected positive against these two

proteins. Results explained that LAM and 38 KDa antigen had the highest

CHAPTER – 1 INTRODUCTION

42

positive reactions in tuberculosis. Patients single positive antibody, two

positive antibody and three positive antibody had sensitivity of 93.1%,

51.3% and 15.6 % and specificity of 81.3 %, 96.6% and 99.3% respectively.