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1

Good afternoon

VIRAL INFECTIONS AND


LABORATORY
DIAGNOSIS

PRESENTED BY: Dr.ESTHER


PRIYADARSHINI.S

CONTENTS
3

INTRODUCTION
CLASSIFICATION OF VIRUSES
Herpes simplex infections
Herpangina
Acute lymhonodular pharyngitis
Hand ,foot & mouth disease
Measles
Rubella

Chickenpox
Herpes zoster
Small pox
Mumps
Infectious mononucleosis
Cytomegalovirus
HIV
Hepatitis
Laboratory diagnosis of viral infections
Conclusion
References

INTRODUCTION
5

Virus (Latin word) = Venom or poison

Viruses Submicroscopic entities which reproduce only


within specific living cells

Do not have cellular organization

Lack enzymes necessary for protein and nucleic acid


synthesis

Infinite in distribution.

Occupy the twilight zone that separates the living from non living

Demonstration by STANLEY in (1935)- viruses could be crystallined


like chemicals

Geirer and schramm (1956)- viruses were only living chemicals

Recent advances in molecular biology --- smallest living units

Viruses offer the best models for understanding the chemistry of life

Viruses consist of central core of DNA or RNA


surrounded by a capsid made up of protiens or
ensheathed outer envelope made of glycoprotiens
& lipids derived from host cell membranes

CLASSIFICATION
8

Classification of viruses according to the type of nucleic acid by


International Committee on Nomenclature of Viruses of the
International Association of Microbiological societies

RNA
DNA

RNA viruses
DNA viruses

CLASSIFICATION OF MAJOR VIRUS GROUPS &


VIRAL DISEASES
9

RNA VIRUSES

1) orthomyxovirus

influenza

2)Paramyxovirus
3)Rhabdovirus
4)Arenavirus

Measles, mumps
Rabies, hemorrhagic fever
Lymphocytic choriomeningitis, lassa
fever

5)calcivirus
6)Coronavirus
7)Bunyavirus

Upper respiratory tract infection

8)picornavirus

Poliomyelitis, coxsackie
diseases,commoncold, foot and mouth
disease

9)Reovirus

10

10) Togavirus
11)Retrovirus( RNA tumor virus

Rubella, yellow fever, St.Louis


encephalitis

DNA VIRUSES
1) Herpes virus
11

a)Herpes simplex 1

Gingivostomatitis, keratconjunctivitis,
genital and skin lesions

b)Herpes simplex 2

Genital & skin lesions,


keratoconjuntivitis, neonatal
infections, meningitis

c)Varicella zoster virus

Varicella (chicken pox)

d)cytomegalovirus

Cytomegalic inclusion disease

e)Epstein Barr virus

Infectious mononucleosis

f)Human herpes Virus 6

Otitis media, encephalitis

g)Human herpes virus 7

Roseola infantum

h)Human herpes virus 8

Infectious mononucleosis

i)Simian herpes virus

Mucocutaneous lesions, encephalitis

12

2)Pox virus

Small pox, molluscum contagiosum

3)adenovirus

Pharyngoconjunctival fever ,
epidemic keratoconjunctivitis

4)parvovirus
5)iridovirus
6)Papovirus

Human warts or papillomas

VIRUS- HOST INTERACTIONS


13

Virus host interactions may cause different effects


ranging from no apparent cellular damage to rapid cell
destruction

Some viruses (e.g. poliovirus)- cause cell death

Others may cause cellular proliferation or malignant


transformation(oncogenic viruses)

14

1) Cytocidal infectionviruses like enterovirus &


reoviruses kill host cells by inhibition of protein, RNA
and DNA synthesis

2) Cell Transformation- infection with hepatitis B


virus(HBV), hepatitis C virus(HCV),Epstein-Barr virus
& several papilloma viruses---- do not result in cell
death, but leads to cell transformation

Transformed cells divide ----- tumor production

15

3) Latent infections
Herpes simplex and varicella zoster viruses remain
latent in the nerve root ganglion, to be reactivated
periodically in some individuals causing recurrent
lesions
Hepatitis B virus chronic infections which may remain
inapparent for many years
Subacute sclerosing panencephalitis- develops between
1- 10 years after recovery of measles virus infection

16

The most characteristic histological feature in virus


infected cells is the appearance of inclusion bodies

Inclusion bodies are structures with distinct size, shape,


location and staining properties that can be demonstrated
in the virus infected cells under the light microscope

They may be situated in the cytoplasm--- e.g.poxvirus


In the nucleus ---- herpes virus
In both --- measles virus

17

The most characteristic histological feature in virus


infected cells is the appearance of inclusion bodies

Inclusion bodies are structures with distinct size, shape,


location and staining properties that can be demonstrated
in the virus infected cells under the light microscope

They may be situated in the cytoplasm--- e.g.poxvirus


In the nucleus ---- herpes virus
In both --- measles virus

18

Inclusion bodies in molluscum contagiosum (molluscum


bodies) are very large( 20-30m)

Intranuclear inclusion bodies were classified into two types


by Cowdry (1934)

Cowdry type A inclusions are of variable size and granular


appearance ( herpes virus)

Cowdry type B are more circumscribed and often multiple


(adenovirus, poliovirus)

19

Inclusion bodies may be crystalline aggregates of


virions or made up of virus antigens present at the
site of virus synthesis

Some inclusions represent degenerative changes


produced by the virus infection which confer altered
staining properties on the cell

20

Herpes simplex infections


Herpangina
Acute lymhonodular
pharyngitis
Hand, foot & mouth disease
Measles
Rubella

Chickenpox
Herpes zoster
Small pox
Mumps
Infectious mononucleosis
Cytomegalovirus
HIV
Hepatitis

HUMAN HERPES VIRUSES


21

Herpes comes from the ancient Greek word meaning to CREEP


or CRAWL

There are 80 known herpesviruses, and at least eight of them


are known to cause infections in humans

These include herpes simplex virus (HSV)1 and 2, varicella


zoster virus (VZV), cytomegalovirus (CMV), Epstein-Barr virus
(EBV), human herpes virus 6 (HHV-6), human herpes virus 7
(HHV-7), and human herpes virus 8 (HHV-8)

Dent Clin N Am 49 (2005) 1529

HERPES SIMPLEX
22

Acute herpetic gingivostomatitis, Herpes labialis, Fever


blisters, Cold sores

Acute infectious disease --- probably the most common viral


disease affecting man

Tissues preferentially involved by HSV often referred to as


herpes hominis--- derived from ectoderm

Skin, mucous membranes, eyes and the central nervous system

23

Herpes viruses contain four layers:


An inner core of double-stranded DNA
A protein capsid
tegument
lipid envelope containing glycoproteins derived from
the nuclear membrane of host cells

24

There are two immunologically different types of HSV Type1


and Type 2

They differ antigenically and biologically


But share 50% of nucleotide sequence

These subtypes can be distinguished serologically or by


restriction endonuclease analysis of DNA

State of latency & reactivation --- common in many viral


infections, especially the herpes group

25

Incubation period is 1-26 days and can occur


through out the year

Transmission is mainly through close contact,


kissing, sharing of glasses etc

PATHOGENESIS
26

Mucosal surfaces & abraded skin favors entry of virus


& initiation of replications in cells of epidermis &
dermis.

Initial or primary infection asymptomatic.

Once virus gains its entry, it enters


sensory, autonomic nerve endings & remain latent in
ganglia

27

28

Virus replication occurs & spreads to other skin &


mucosal surfaces by centrifugal distribution of virions
through peripheral nerves

Disease manifests as charecteristic vesicles containing


desquamated cells, multinucleated giant cells & free
virus with edema fluid

HSV -1 - face ,lips ,oral cavity & upper half of body


HSV -2- genitalis & skin of lower half of body.

29

Primary infection resolves & virus cannot be recovered


from ganglia.

Humoral & cell-mediated immunity responsible for


clinical manifestation, latency & recurrence of disease

30

Two types of infection with herpes simplex occur


Primary infection- person who does not have
circulating antibodies
Recurrent infections persons who have such
antibodies
Impossible to differentiate clinically between primary
and recurrent infection
Primary infection is accompanied more frequently by
systemic manifestations and is fatal

FORMS OF HSV
31

Herpes genitalis
HSV -2
Uterine cervix,vagina,vulva & penis.
Now often termed as new epidemic venereal diseasesince it is transmitted through sexual contact
More virulent than HSV-1

32

Herpes meningoencephalitis
Sudden fever & symptoms of increased intracranial pressure.
Paralysis of muscle groups, convulsions and death may occur
Herpes conjunctivitis /Keratoconjunctivitis
Swelling & congestion of palpebral conjunctiva.
Keratitis & corneal ulceration.
Herpetic vesicles of eyelids are seen which heal rapidly.

33

Herpetic eczema /Kaposis varicelliform eruption

Epidermal form of herpetic infection superimposed


upon pre-existing eczema.
Diffuse vesicular lesions of skin.
Children - high fever with typical umbilicated
vesicles.

34

Disseminated herpes simplex of new born


Newborn acquires infection during passage through birth canal
of mother.
Manifestation - by 4-7th day
Few die on 9-12th day.
Surviving infants show neurological involvement
Herpetic whitlow
In fingers due to autoinoculation
Herpes gladiatarum --- In wrestlers

PRIMARY HERPETIC STOMATITIS


35

Common children & young adults

Transmission droplet spread & contact with lesions

Affects children and young adults

It rarely occurs below the age of six months--because of presence of circulating antibodies in the
infant derived from the mother

36

In children disease is primary attack--- development of fever


,irritability,headache , pain upon swallowing & regional
lymphadenopathy

With in few days mouth becomes painful & gingiva appears


erythematous & edematous.

Yellowish,fluid filled vesicles develop which rupture to


form shallow, ragged, painful ulcers covered by gray
membrane & surrounded by erythematous halo

37

Lips, buccal mucosa, palate,pharynx may also be


involved

38

It is important to recognize that the gingival


inflammation precedes the formation of the ulcers by
few days

Ulcers vary considerably in size - from tiny lesions to measuring several millimeters to even a centimeter in
diameter

Heal spontaneously- 7-14 days


leave no scar

39

HSV does not remain latent at the site of the


original infection
Instead , reaches the nerve ganglia supplying the
affected area--- remain latent there until
reactivated
Usual ganglia involved are trigeminal- HSV-1
Lumbosacral --- HSV-2

HISTOLOGICAL FEATURES
40

Herpetic vesicle is an intraepithelial blister filled with


fluid.
Infected cells are swollen & have pale eosinophilic
cytoplasm & large vesicular nuclei---Ballooning
degeneration
Acantholytic epithelial cells are called as Tzanck cells

41

intranuclear inclusions
Lipschutz bodies

Eosinophilic, ovoid, homogenous


structures within the nucleus,
which tend to displace the
nucleolus and nuclear chromatin
peripherally

Displacement of chromatin often


shows peri- inclusion halo

42

Multinucleated infected epithelial cells are formed


when fusion occurs between adjacent cells

CT is infiltrated by inflammatory cells.

When vesicle ruptures,surface of tissue is covered by


exudate - fibrin,PMNLs & degenerated cells.

LABORATORY DIAGNOSIS
43

Diagnosed both clinically and laboratory procedures

History and physical examination is sufficient to make


a diagnosis of HSV infection

GOLD STANDARD viral culture

The goal of virus isolation is to observe cytopathic


effects of the cells inoculated with virus.

44

Cytopathic effects are the degenerative changes that cells undergo


when infected with virus

The rate at which cytopathic effects develop depends on the type of


host cell, the type of virus, and the concentration of virus

When viewed at high power using light microscopy, virally infected


cells demonstrate multinucleated giant cells,syncitium, and
ballooning degeneration

Dent Clin N Am 49 (2005) 1529

45

Cytology smears
A smear taken of epithelial cells at the base of a suspected lesion
studied to determine if epithelial cells show changes consistent
with HSV infection.

The most common stain used is Giemsa

When a Pap stain has been performed, eosinophilic intranuclear


viral inclusion bodies (Lipschutz or Cowdry type A) can be seen.

46

Tzanck test
Tzanck preparation is a rapid test done to diagnose
infections caused by herpesviruses.
Cells are examined under a microscope for signs of
infection
The Tzanck preparation is done by smearing cells taken
from a fresh blister or ulcer onto a microscope slide.

47

The cells are stained with a special stain, such as Wright's stain,
and then examined under a microscope for characteristic
changes caused by a herpesvirus
Herpes causes giant cells with multiple nuclei

The background of the cell looks like ground glass and contains
small dark spots called inclusion bodies

Less sensitive --- cannot differentiate between HSV-1 & HSV-2

48

Immunomorphologic tests

The diagnosis of herpesvirus infections can be made more quickly and


accurately by using immunomorphologic techniques

In the direct fluorescent assay (DFA), the specimen is incubated with


fluorescein isothiocyanatelabeled HSV type-specific monoclonal antibody.

The positively infected cells are fluorescent green when examined under a
fluorescent microscope.

Dent Clin N Am 49 (2005) 1529

49

Serologic tests

Serologic tests are completed to detect antibody


formation in a patients blood sample

They are useful in diagnosing a primary HSV infection

A fourfold or greater antibody rise in convalescent


serum is required for the diagnosis of a primary
infection.

50

If serology is used in the diagnosis of suspected


HSV infection, an acute specimen should be
obtained within the initial 3 days of the infection
convalescent specimen should be obtained
approximately 4 weeks later
Because of the delayed humoral response,
antibodies are not present in the acute specimen
but appear during convalescence.

TREATMENT
51

Antiviral drugs - impact on course of disease


Antibiotic therapy prevention of 20 infection.
NSAIDS & anaesthetic gel relieve discomfort

RECURRRENT OR SECONDARY HERPETIC


LABIALIS & STOMATITIS
52

Usually seen in adult patients

Manifests itself clinically as accentuated form of the primary disease

Recurrent form of disease is associated with trauma, fatigue, menstruation,


pregnancy, upper respiratory tract infection, emotional upset, exposure to sunlight

Occurs -every month once in a year or less

Lesions develop at site of primary inoculation or adjacent area supplied by


involved ganglia

It may develop on lips or intraorally

53

Prodromal symptoms ---burning or


tingling sensation & feeling of
tautness,swelling or soreness

Gray /white vesicles rupture


quickly,leaving small red ulceration

On lips these ruptured vesicles


become covered by brownish crust

Lesions heal within 7-10 days &


leaves no scar

54

Weathers and Griffin--- that the recurrent intra


oral herpetic lesions almost invariable develop on
oral mucosa that is tightly bound to periosteum

LABORATORY DIAGNOSIS
55

Immunofluorescent staining of smears--- with


fluorescein labeled HSV antibody

Serological assays - Complement fixation assay


Radio immunoassay
ELISA

56

Treatment
Acyclovir
Vidarbine
Idoxuridine

HERPANGINA
57

Aphthous pharyngitis/vesicular pharyngitis

Etiology Coxsackie group viruses with types 1,6,8,10


or 22

Transmission Ingestion,direct contact, droplet spread

It is chiefly a summer disease ----many children may


actually harbor the virus without exhibiting clinical
manifestation of the disease

CLINICAL FEATURES
58

Incubation period - 2-10 days

Common - young children

It begins with sore throat, cough,


rhinorrhea, low grade fever, headache

Small vesicles which rupture to form


ulcers, showing a gray base & inflamed
periphery on anterior faucial pillars

59

Ulcers are not extremely painful though dysphagia is


seen
Ulcers heal within 7-10 days
A permanent immunity to infecting strain develops
rapidly & have neutralizing Abs against numerous
strains.

LABORATORY FINDINGS
60

Coxsackie virus can be isolated in mice by


inoculation of scrapings from throat lesions or
stool specimens of nearly all patients

Although there are distinct immunologic


differences between various strains of herpangina
virus, animal inoculation of any type produces the
same manifestations

61

Treatment
No treatment is necessary as disease - self limiting
& have few complications

ACUTE LYMPHONODULAR PHARYNGITIS


62

Etiology Coxsackie virus A 10.

Incubation period - 2-10 days.

Common - children & young adults.

Chief complaints consist of sore throat, elevation


of temp -100O-1050 F, mild headache & anorexia.

63

Symptomatic course - 4-14 days

Local oral lesions heal - 6-10 days but a residual


ring of fading erythema seen for several days

Estimated incubation period is 2-10 days

ORAL MANIFESTATIONS
64

Lesions are raised,discrete,whitish or yellowish to


dark pink solid papules or nodules,surrounded by
narrow zone of erythema.
Lesion are not vesicular and do not ulcerate
Lesions appear on uvula, soft palate, anterior pillars
& posterior oropharynx.

LABORATORY FINDINGS
65

Isolation of coxsackie A 10 virus can be established in


suckling mice by inoculation of throat swab or fecal
material

Serological evidence of infection by the virus is also


positive

66

Histologic features
Papules or nodules consist of hyperplastic
lymphoid aggregates.
In some cases, intranuclear inclusion bodies are
seen.
Treatment
No treatment is necessary as disease is self
limiting

HAND,FOOT & MOUTH DISEASE


67

Etiology Coxsackie A 16
Less frequently by A5 A6

Common - Young children

Appearance of maculopapular ,
exanthematous & vesicular lesions of skin
particularly involving the hands, feet, legs
and arms

Patients also commonly manifest anorexia


,low grade fever, coryza, lymphadenopathy,
diarrhea, nausea and vomiting

ORAL MANIFESTATIONS
68

Sore mouth and refusal to eat is one


of the most common findings in this
disease

Due to small, multiple vesicular and


ulcerative oral lesions that are more
numerous than in herpangina

Common sites Hard palate,tongue


& buccal mucosa

Tongue becomes red & edematous

LABORATORY FINDINGS
69

Intracytoplasmic viral inclusions seen in vesicular


scrapings.
Viral isolates obtained from rectal or throat swabs
from vesicular fluid itself
Remarkable rise in the acute or convalescent serum
antibody titre to coxsackie A16
TREATMENT
No specific treatment

FOOT AND MOUTH DISEASE


70

Aphthous fever, Hoof and mouth disease, Epizootic stomatitis

Transmission --- Use of milk from affected animals or through


handling of tissues from these animals

Clinical features

Fever ,nausea ,vomiting, malaise & appearance of ulcerative


lesions of oral mucosa & pharynx

Development of vesicles on skin also occurs.


Common on palms & soles.

MEASLES
71

Rubeola,Morbilli.
Acute, contagious, dermatropic viral infection
primarily affecting children
Etiology - Paramyxovirus (RNA virus)
Incubation period - 8-12 days
Transmission - respiratory secretions or direct
contact of droplets.

PATHOGENESIS
72

Upon invasion of respiratory epithelium ,it reaches RES


through blood stream & thereby infects skin, respiratory
tract & other organs

Invasion of T-lymphocytes & increased levels of


suppressive cytokines - suppression of cellular immunity

Viremia develops, but specific Abs are not developed


before onset of rash.

CLINICAL FEATURES
73

Measles arise in the winter and spread through respiratory


droplets
Incubation period is 10-12 days
Affected individuals are infectious from 2 days before becoming
symptomatic and until 4 days after the appearance of the
associated rash
Virus is associated with significant lymphoid hyperplasia --involves sites such as tonsils, adenoids and peyers patch
Gaint cell infiltration is noted in various tissues along with
vasculits responsible for characteristic rash

74

Three stages of infection--- each stage lasting for 3


days justifying the designation --- NINE- DAY
MEASLES

First 3 days are dominated by three Cs--- coryza


(runny nose)
Cough
Conjuctivitis
Fever typically accompanies these symptoms

75

During the initial stage most distinctive oral


manifestation--- Kopliks spots
Multiple areas of mucosal erythema on buccal
and labial mucosa
Within these areas are numerous small, blue white
macules

76

Second stage begins---fever continues, kopliks


spots fade and a maculopapular and
erythematous rash begins
Face is involved first, with eventual downspread
to the trunk and extremities
Diffuse Maculopapular eruption is formed, which
tends to blanch on pressure

77

Third stage --- fever ends


Rash begins to fade and demonstrates similar downward
progression with replacement by a brown pigmentary staining

Common complications in children--- otitis media, pneumonia,


persistent bronchitis and diarrhea

Delayed complication termed--- subacute sclerosing


panencephelitis arises as late as 11 years after the initial infection

Degenerative disorder of CNS--- personality changes, seizeurs,


coma and death

HISTOPATHOLOGICAL FEATURES
78

Initially koplik spots represent areas of focal


hyperparakeratosis --- underlying epithelium exhibits
spongiosis, intercellular edema, dyskeratosis, and
epithelial syncitial gaint cells

Number of nuclei within these gaint cells ranges from


three to more than 25

Close examination of epithelial cells reveals pink-staining


inclusions in the nuclei or less common in the cytoplasm

79

On electron microscopy, the inclusions have been


shown to represent microtubular aggregates
characteristic of the causative paramyxovirus

Spot ages --- epithelium exhibits heavy exocytosis by


neutrophils leading to microabcess formation,
epithelial necrosis and ultimately, ulceration

Examination of epithelium adjacent to the ulceration


will reveal the suggestive syncitial gaint cells

80

Examination of hyperplastic lymphoid tissue during prodomal


stage of measles reveals a similar alteration
In 1931Warthin and Finkeldey separate publications, reported an
unusal finding in the patients who had their tonsils removed
Within the hyperplastic lymphoid tissue, there were numerous
multinucleated giant lymphocytes termed as WARTHINFINKELDEY giant cells

CONTROL MEASURES
81

MMR vaccine -5ml is given subcutaneously.


1st dose 9 months
2nd dose 15 -18 months

RUBELLA/GERMAN MEASLES
82

Mild viral illness that is produced by a virus in the


family Togavirus, genus Rubivirus

Gretaest importance of this infection lies not in the


effects on those who contract the acute illness, but in its
capacity to induce birth defects in the developing fetus

Infection occurs primarily in winter and spring

Contracted through respiratory droplets

83

Incubation time --- 14-21 days

Infected patients are contagious from 1 week before


the exanthem to 5 days after the development of rash

Infants with congenital infection may release virus


for upto 1 year

CLINICAL FEATURES
84

Infection are asymptomatic

Frequency of symptoms greater in adolescents and adults

Prodomal symptoms before exanthem --- fever, headache,


coryza, malaise, anorexia,myalgia, mild conjunctivitis and
lymphadenopathy

Lymphadenopathy --- suboccipital, postauricular and


cervical chains

85

Exanthematous rash --- first sign of infection

Begins on face and neck, spreads to entire body within 3 days

Rash forms discrete pink macules, then papules and finally


fades with flaky desquamation

Exhibits facial clearing before the rash spreads to the lower


body areas

Rash is resolved in 3 days--- three- day measles

ORAL MANIFESTATIONS
86

Oral lesions known as FORCHHEIMERS SIGN are


seen

Consist of small,discrete, dark red papules that


develop on the soft palate and may extend onto hard
palate

Enanthem arises simulaneously with rash, become


evident in about 6 hrs after the first symptom and not
lasting longer than 12-14 hours

DIAGNOSIS
87

Viral culture is possible


Serological analysis is the mainstay of diagnosis

SMALL POX / VARIOLA


88

Small pox is an acute viral disease which before the discovery of


vaccination by Jenner was epidemic in nature and accounted for
millions of deaths

On Dec 9,1979, the WHO Global Commission for the


certification of small pox Eradication declared

1)small pox eradication has been achieved through out the world

2) there is no evidence that small pox will return as an endemic


disease

89

This was officially endorsed by the World Health


assembly meeting at Geneva on May 8,1980, when in
resolution 33.3, it declares solemnly that the world
and all its people won freedom from small pox
an unprecedented achievement in the history of
public health

CLINICAL FEATURES
90

Incubation period 7-10 days

Skin lesions begin as small macules & papules on


face spreading to other parts.

Papules

Pustules are small, elevated and yellowish green with


an inflamed border

Vesicles

Pustules

91

Pustules - secondarily infected - become


hemorrhagic

Desquamation healing phase

Complication - severe pitting of skin, formation of


abscess ,septicemia

MOLLUSCUM CONTAGIOSUM
92

Disease caused by a virus of pox group


Lesions which occur only occur on skin or mucosal
surfaces
Often considered tumor like in nature---- typical
localized epithelial proliferation caused by the virus

CLINICAL FEATURES
93

More common in children

Manifests itself as single or more frequently multiple


discrete elevated nodules--- occuring on the arms and
legs, trunk and face particularly the eyelids

Lesions are hemispheric in shape --- about 5mm in


diameter with a central umblication which may be
keratinized and are normal or slightly red in colour

94

Apperas to be spread by autoinoculation

Lack of inflammation and necrosis differentiates these


proliferative lesions from other pox virus lesions

HIV individuals are more prone to these lesions

Linear distribution of these lesions suggests that


autoinoculation of virus occurs due to scratching

HISTOPATHOLOGICAL FEATURES
95

Appears as a localized lobular proliferation of surface stratified


squamous epithelium

Central portion of each lobule is filled with bloated keratinocytes


that contain large, intranuclear basophilic viral inclusions called
molluscum bodies or Handerson- Paterson bodies

These bodies begin as small eosinophilic structures in cells just


above the basal layer

Central crater --- stratum corneum cells disintegrate to release


their molluscum bodies

CONDYLOMA ACUMINATUM
96

Is an infectious disease caused by a virus which belongs to


same group of Human papilloma viruses

It is caused by HPV type 6,a11, a30, b42, 43,45,46b,51,54,55 &


70

The virus of anal, genital and presumably oral condyloma


acuminatum is HPV6

Transmission close contact with infected persons,


autoinoculation,and orogenital sexual practice

CLINICAL FEATURES
97

Oral lesions frequently occur on labial mucosa,soft palate and


lingual frenum
Typical condyloma appears as sessile, pink,well demarcated,
non tender exophytic mass with short blunted surface
projections
Larger than papilloma
Average lesional size is 1.0-1.5 cm

HISTOPATHOLOGICAL FEATURES
98

Appears as benign proliferation of


acanthotic startified squamous epithelium
with mildly keratotic papillary surface
projections

Thin connective tissue cores support the


papillary epithelial projections , which are
more blunted and broader than those of
squamous papilloma

Epithelium is mature and differentiated, but


prickle cells often demonstrate pyknotic,
crinkled nuclei surrounded by clear
zones( koilocytes)

99

Ultra structural examination reveals virions within


the cytoplasm or nuclei of koilocytes

Virons can also be demonstrated by


immunohistochemical analysis and PCR techniques

CHICKENPOX(VARICELLA)
100

Chickenpox acute,ubiquitous,contagious disease in children

Characterized by exanthematous vesicular rash

Common - winter & spring months.

Etiology - Varicella zoster virus.

Incubation period - 2 weeks

Mode of transmission - air borne droplets, direct contact with


lesions.

CLINICAL FEATURES
101

Prodromal symptoms - headache, nasopharyngitis & anorexia


followed by maculopapular or vesicular eruptions of skin &
low-grade fever
Begin on trunk & spread to face & extremities.
They occur in successive crops so that many vesicles in different
stages of formation or resorption are found

102

Lesions of skin rupture,form superficial crust & heal


by desquamation.

Secondary infection formation of pustules, which


leave small pitting scars upon healing.

ORAL MANIFESTATIONS
103

Small blister like lesions involve oral mucosa chiefly


buccal mucosa, gingiva & palate pharyngeal mucosa.
Slightly raised vesicles with surrounding erythema
rupture & form ulcers
These lesions are relatively painless

Complications
104

Secondary infection by staphylococcucs/


streptococcus
Encephalitis
Myocarditis
Nephritis
Arthritis

HERPES ZOSTER
105

Shingles/Zona
Acute infectious viral disease - extremely painful and
incapacitating nature - characterized by inflammation of
dorsal root ganglia or extra- medullary cranial nerve ganglia

Associated with vesicular eruptions of skin and mucous


membrane in areas supplied by affected sensory nerves

Reactivation of latent V-Z virus acquired during previous


attack of chickenpox

CLINICAL FEATURES
106

Common Adult life

Affects Males & females with equal


frequency

Fever,malaise, pain & tenderness unilaterally along course of sensory nerves

Linear papular or vesicular eruption of


skin.

After rupture of vesicles healing


commences.

ORAL MANIFESTATIONS
107

Herpes zoster involves face by infection


of 3 branches of trigeminal nerve.

Painful vesicles buccal mucosa, tongue,


uvula, pharynx & larynx

Characteristic feature is unilaterality

Vesicles rupture to leave areas of erosion.

Lesions extend upto midline & stop


abruptly

JAMES RAMSAY HUNTS SYNDROME


108

Special form of zoster infection of geniculate ganglion


Facial paralysis

Pain of external auditory meatus & Pinna of ear

Vesicular eruptions in oral cavity & oropharynx with


hoarseness ,tinnitus, vertigo

109

Postherpetic neuralgia (PHN) is a potential consequence of HZ


resulting

from scarring of the involved nerve during infection PHN is a


painful, sometimes debilitating condition that can last months to
years after the lesions are healed.

The incidence of PHN is increased in patients older than 50


years

110

Diagnosis:
Recognized by its characteristic distribution of lesions
Skin & oral lesions identified by cytologic smear &
finding of Tzanck cells.
To differentiate between herpes zoster and simplex--fluroscent antibody staining technique, seroliogical
diagnosis and viral culture can be done

MUMPS
111

Epidemic parotitis

Acute contagious viral infection Characterized chiefly


by unilateral or bilateral swelling of the salivary
glands, usually parotid

It may also involve meninges, pancreas and gonads

Disease of childhood , may also affects adults

Incubation period - two to three weeks.

Transmission -

respiratory route.

PATHOGENESIS
112

Once transmitted through droplet nuclei or saliva , it starts


replicating in the respiratory epithelium.

It spreads to local lymph nodes develops viremia.

Affected area - perivascular and interstitial mononuclear cell


infiltrates with edema.

Necrosis of acinar and epithelial duct cells seen in salivary glands.

CLINICAL FEATURES
113

Disease is usually preceeded by the onset of headache, chills,


moderate fever,vomiting and pain below the ear

Firm, rubbery or elastic swelling of the salivary glands elevating the ear -lasts for one week.

Pain and tenderness - severe during the rapid phase of parotid


enlargement .

swelling reaches its maximum -

3 days

114

It remains at its peak for 2 to 3 days and then


gradually subsides.

Presternal edema may be seen due to pressure on


lymphatics in the neck.

Papilla of the opening of the parotid duct on the


buccal mucosa is often puffy and reddened.

DIAGNOSIS
115

Virus can be isolated from saliva and throat swab 2


days before or 7 days after the onset of parotitis
Also be confirmed by the complement fixation test
or ELISA .
Serum amylase is elevated in both parotitis and
pancreatitis.

COMPLICATIONS
116

When mumps involves the adult male- orchitis


---complete sterility

Involvement of the pancreas - acute pancreatitis

Meningoencephalitis, deafness, and mastitis are


occasional complications.

Prevention
117

MMR vaccine
1st dose 9 months
2nd dose- 15 -18 months

INFECTIOUS MONONUCLEOSIS
118

GLANDULAR FEVER , KISSING DISEASE

Etiology ---Epstein-Barr virus

Children are infected through contaminated saliva on fingers, toys,


or other objects.

Adults are infected through direct salivary transfer, such as shared


straws or kissing, hence, the name "kissing disease

Glandular fever was first used by the German physicians in 1889

CLINICAL FEATURES
119

Most EBV infections children- asymptomatic.

In children younger than 4 years of age -fever,


lymphadenopathy, pharyngitis, hepatosplenomegaly,
and rhinitis or cough.

Children older than 4 years --- exhibit lower


prevalence of hepatospleenomegaly, rhinitis and cough

120

Most young adults experience fever, lymphadenopathy,


pharyngitis, and tonsillitis

In classic infectious mononucleosis in a young adult, prodomal


fatigue, malaise , anorexia occur upto 2 weeks before the
development of pyrexia

Body temperature may reach 104F and lasts from 2-14 days

Cervical lymphnodes are usually the first to exhibit enlargement


followed by the nodes of axilla and groin

ORAL MANIFESTATIONS
121

No specific oral manifestation


Consisted chiefly of acute gingivitis & stomatitis
Apperance of gray areas membranes in various areas
Palatal petechiae & oral ulcers
Edema of soft palate and uvula has also been reported

LABORATORY FINDINGS
122

White blood cells count is increased, with the differential count showing
relative lympocytosis

Atypical lymphocytes are present in the peripheral blood

Atypical cells are lymphoblasts derived from T-cells reactivate to infection

Oval, horse shoe shaped or indented nuclei with dense irregular nuclear
chromatin and a basophilic foamy cytoplasm

Classic serologic finding --- presence of Paul-Bunell heterophile


antibodies

Paul- Bunnell test


123

Heterophile antibodies appear in the serum of the patient


These are IgM antibodies elicited by EBV infection
Seen during acute phase of infection
Their titre decrease rapidly after fourth week of infection
These antibodies agglutinate sheep erythrocytes

PROCUDURE
124

Inactivated serum (560C for 30 min) in doubling dilutions is


mixed with equal volumes of 1% sheep erythrocyte
suspension
Tubes are incubated at 37 degree centigrade for 4hrs and
examined for agglutination
A titre of 100 or above is suggestive of infectious
mononucleosis
Normal titres of agglutinins in human blood against sheep
red blood cells does not exceed 1:8
In infectious mononucleosis --- titre rise to 1:4069
Referred to as POSITIVE PAUL BUNNEL TEST

EBV-SPECIFIC ANTIBODIES
125

Specific antibodies against EBV viral capsid


antigen(VCA)
IgM antibody to VCA --- acute infections
IgG antibody to VCA
Later in the course of the disease --- increase in IgM
(VCA) +increase in IgG (VCA)+ increase in IgG( EBNA
antibodies)--- appears after 1-2 months and persists
through out life
Suggests previous exposure to the antigen

CYTOMEGALOVIRUS
126

CMV after initial infection latent in salivary


gland cells, endothelium, macrophages,
lymphocytes.

In infants virus is contracted through the placenta,


during delivery or during breast feeding

127

Most of CMV infections are asymptomatic.

In clinically evident neonatal infection, infants appears


ill within a few days.

Typical features - hepatosplenomegaly, extra medullary


cutaneous erythropoiesis and thrombocytopenia .

Encephalitis - severe mental and motor retardation.

128

In a young adult, prodromal fatigue, malaise, and


anorexia occur up to 2 weeks before the
development of pyrexia.

The temperature may reach 104 F and lasts from


2 to 14 days.

129

Petechiae on the hard or soft palate are present.

The petechiae are transient and usually disappear


within 24 to 48 hours.

HISLOPATHOLOGIC FEATURES
130

Biopsy specimens - demonstrate changes within the


vascular endothelial cells.
Scattered infected cells are extremely swollen,
showing both intracytoplasmic and intranuclear
inclusions and prominent nucleoli.
This enlarged cell -called -"owl eye" cell.

DIAGNOSIS
131

Detection of viral antigen by immunohistochemistry


Polymerization chain reaction
Demonstration of viral antibody titres
Viral culture

ACQUIRED IMMUNODEFICIENCY
SYNDROME (AIDS)
132

Etiology

- Human Immunodeficiency Virus (HIV)

HIV is a retro virus having the nucleic acid in the form of


RNA

During replication the RNA must be changed into DNA


utilizing the enzyme reverse transcriptase which enables
integration to the host DNA.

133

Two genetically distinct populations of viruses


known to cause AIDS are HIV-1 and HIV-2.

HIV-1 - central Africa

HIV-2 -

West Africa and India.

134

HIV was first isloated by Luc Montaginer from Pasteur


Institute, Paris in 1983 from a West African patient
suffering from persistent generalised
lymphadenopathy.

In india the first case of HIV/AIDS was detected in the


year 1986 in chennai. Since then the virus has moved
across all states in India and today we see cases of
HIV/AIDS across the country

In A.P. First death reported in 1987

HIGH RISK DISTRICTS IN ANDHRA


PRADESH

Vishakhapatnam
East Godavari
Warangal
Medak
Chittoor
Kurnool
13
5

MODES OF HIV/AIDS TRANSMISSION


136

137

AIDS is only the last stage in wide spectrum of clicnical


features in HIV infection.
Classification system for HIV infection(CDC,USA)
Group 1- Acute infection
Group II - Asymptomatic infection
Group III Persistent Generalised Lypmhadenopathy
Group IV AIDS Related Complex

Stage 1 - Primary

Short, flu-like illness - occurs one to six weeks


after infection
no symptoms at all
Infected person can infect other people

Stage 2 Asymptomatic

Lasts for an average of ten years


This stage is free from symptoms
There may be swollen glands
The level of HIV in the blood drops to very low levels
HIV antibodies are detectable in the blood

Stage 3 - Symptomatic

The symptoms are mild


The immune system deteriorates
emergence of opportunistic infections and
cancers

Stage 4 - HIV AIDS

The immune system


weakens

The illnesses become


more severe leading to
an AIDS diagnosis

142

143

When HIV is transmitted - fusion with CD4


cells occurs rapidly, and within a few days the
virus migrates to regional lymph nodes, and
then enters the circulation system.

This results in widespread dissemination to the


brain and the lymphatic system, causing
'primary HIV infection' or 'seroconversion
syndrome.'

144

After infection, there is a rapid rise in plasma viremia,


often to levels in excess of 1 million viral RNA
molecules per ml

After the initial rise in plasma viremia, there is a


marked reduction from the peak viremia to a steadystate level of viral replication.

145

Within 6-12 weeks, antibodies to HIV are detectable in the


blood.

At this time, patients are confirmed as HIV positive.

The time between viral infection and seroconversion is referred


to as the 'window period.'

146

CLINICAL FEATURES

Patients - asymptomatic CD4 < 500cells/mm3

AIDS

- diagnosed

- CD4 < 200cells/mm 3

ORAL MANIFESTATIONS IN HIV


147

Oral lesions are often clearly visible and several


can be diagnosed accurately on clinical features
alone
Oral lesions --- important as they not only affect
the quality of life
Useful markers for disease progression and
immunocompression

REVISED CLASSIFICATION IN 1993 BY CDC


148

Group 1: Lesions strongly associated with HIV infection


Candidiasis
Erythematous ,Pseudomembranous
Hairy leukoplakia
Kaposi's sarcoma
Non-Hodgkin's lymphoma
Periodontal disease
Linear gingival erythema ,Necrotizing ulcerative

gingivitis ,Necrotizing ulcerative periodontitis

149

Group 2: Lesions less commonly associated with HIV


infection

Bacterial infections
Mycobacterium avium intercellulare,
Mycobacterium tuberculosis
Melanotic hyperpigmentation
Necrotizing ulcerative stomatitis

150

Group 3:

Lesions seen in HIV infection

Bacterial infections
Actinomyces israelii
Escherichia coli
Klebsiella pneumoniae
Cat-scratch disease

Drug reactions (ulcerative, erythema multi-forme,


Lichenoid reaction, toxic epidermolysis)

151

Fungal infections other than candidiasis


Cryptococcus neoformans
Geotrictum candidum
Histoplasma capsulatum
Miscoraceae (zygomycosis)
Aspergillus flavus

152

Neurologic disturbances
Facial palsy,Trigeminal neuralgia
Recurrent apthous stomatitis
Viral infections CMV, Molluscum contagiosum

ORAL CANDIDIASIS
153

Pseudomembranous candidiasis
White to yellowish white plaques - easily scraped off,
exposing red areas.
Lesions - extensive, involving > one site in the oral
cavity.
Involve the oropharynx and eosophagus

154

Erythematous candidiasis
Clinically seen as red lesions - located on the dorsum
of the tongue, palate, and buccal mucosa.
Tongue lesions are also referred to as central papillary
atrophy.

HYPERPLASTIC CANDIDIASIS
155

White plaques - cannot be removed


by scraping.

Diagnosis can be confirmed by


biopsy, which demonstrates the
fungal hyphae in the keratinized
layers of the epithelium.

Can be differentiated from other oral


white lesions as these respond to
topical or systemic antifungal therapy

ANGULAR CHEILITIS
156

Erythema or fissuring or scaling of the angles of


the mouth .
Microbiologically - due to mixed infection of
Candida albicans and staphylococcus aureus .

PERIODONTAL LESIONS
157

Periodontitis in HIV-positive patients


Age
Smoking
Viral load
Microorganisms -Fusobacterium nucleatum,
Prevotella intermedia,
Actinobacillus actinomycetemcomitans

LINEAR GINGIVAL ERYTHEMA (LGE)

Is a non-plaque induced
gingivitis.

Erythematous band of the


marginal gingiva with either
diffuse or punctuate erythema
of attached gingiva.

158

NECROTIZING PERIODONTAL DISEASES


159

Subclassified as

Necrotizing ulcerative gingivitis (NUG)


Necrotizing ulcerative periodorititis (NUP)
Necrotizing stomatitis (NS).

NECROTIZING ULCERATIVE GINGIVITIS

Involves destruction of one


or more interdental
papillae and is limited to
the marginal gingiva

160

NECROTIZING ULCERATIVE PERIODONTITIS

NUP extends beyond the


papillae and marginal,
gingiva causing the loss of
the periodontal! attachment,
and possibly exposing the!
bone.

161

NECROTIZING STOMATITIS

When the necrosis extends


beyond: the periodontium
into the mucosa & osseous
tissue, it results in NS

162

Herpes Simplex Virus Infection

163

Intraoral lesions on the gingiva are referred to as


acute herpetic gingivostomatitis.
It involve the palate, pharynx, and tonsils.
The lesions - numerous pinhead sized vesicles collapse to form small ulcers exhibiting a red base
covered with yellow fibrin.

HERPES ZOSTER
164

It is a recurrent viral infection in HlV patients .

Herpes zoster begins as a unilateral cluster of


vesicles and ulcers.

ORAL HAIRY LEUKOPLAKIA


165

It appears bilaterally on the


lateral borders of the tongue

painless, faint white vertical


streaks or thickened & furrowed
areas with a shaggy keratotic
surface with vertical striations

imparting a corrugated
appearance.

KAPOSI'S SARCOMA
166

Kaposi's sarcoma (KS) is a


multifocal neoplasm of vascular
endothelial origin.
Human herpes virus type 8 is
involved in the pathogenesis of
KS.
Common - palate, gingiva,
tongue, and oropharynx or the
skin

167

Clinical appearance of oral KS macular, nodular, or raised and


ulcerated, the color of which can
range from red to purple.

Early lesions tend to be flat, red


and asymptomatic, with the color
becoming darker as the lesion age.

APHTHOUS ULCERS
168

Aphthous ulcers present as recurrent,


round, shallow, painful ulcers of
variable size and duration that are
typically found on nonkeratinized oral
mucosa.

They usually measure more than 2 cm


in diameter with regular borders

ORAL SQUAMOUS CELL CARCINOMA (OSCC)


169

OSCC has reported in HIV/AIDS patients at


a younger age itself.

It is due to Impaired immunosurveillance,


and an increased chance of human
papilloma virus infection .

MOLLUSCUM CONTAGIOSUM
170

It is an infection of the skin, caused by a pox


virus.

It presents as shiny, white, and hemispherical


skin-colored dome-shaped papules that often
demonstrate a central depressed crater.

THROMBOCYTOPENIC PURPURA (TP)


171

TP is a hematologic disorder characterized by a


decreased number of circulating blood platelets
(less than 150,000 platelets/mm3 of blood).

The reduction may be due to reduced production,


increased destruction and sequestration subsequent
to spleenomegaly.

172

HIV TP is characterized by reduced


production of platelets due to drugs,
malnutrition, immunological alterations,
microbial invasion, or due to the course of
HIV disease.

Oral TP is characterized by pinpoint


petechiae following minor trauma and
even mastication.

Spontaneous gingival hemorrhage is


common.

NON-HODGKIN'S LYMPHOMA
(NHL)
173

Second most common cancer associated with HIV.

Present as a large, painful, ulcerated MASS on the


palate or gingival tissues or tongue.

HYPERPIGMENTATION
174

Oral hyperpigmentation in HIV infection due to


several medications - ketaconazole and
zidovudine.

Diagnosis - recent onset of brown to brownishblack intraoral focal or diffuse macules.

LABORATORY INVESTIGATIONS OF HIV INFECTION


175

Recommended persons for HIV testing include


176

Have a history of identifiable risks.


Received transfusion of blood or blood products.
Are planning marriage
Pregnant women
Are admitted to hospital
Donors of blood, sperms, organs
Consider themselves at risk.

SPECIFIC TESTS
177

Demonstration of HIV, its antigens or antibodies

Virus isolation
Antigen detection
Antibody detection ELISA
Western blot test
Polymerase chain reaction

VIRUS ISOLATION
178

It can be isolated from CD4 lymphocytes of


peripheral blood, bone marrow and serum.

Virus titres are high early in infection before


antibodies appear.

During asymptomatic infection, virus titre is low


and may not be detectable, but when clinical
disease sets in, the titre rises once again.

PROCEDURE
179

Pts Peripheral Blood Mononuclear Cells + uninfected PBMC


treated with IL-2.
Growth of virus can be detected in 7 - 32 days ,by measuring RT
activity or the production of viral p24 core Ag by enzyme immuno
assay of culture medium.
Detection of RT activity - retrovirus,
Ag assays are specific for HIV.

USES
180

Directly detects the virus and hence is highly specific.


The virus can be cultured from pts specimen at any time in the
course of the disease.
positive even during the window period and during the terminal
phase when the viral load is high and when the ELISA may be
negative

Disadvantage
Not suitable as a routine diagnostic test.
Risky
With the availability of PCR, HIV culture is not done routinely.

ANTIGEN DETECTION
181

The major core antigen P24 is the earliest virus


markers to appear in blood and is the one tested for .
Free P24 antigen - absent during the long
asymptomatic phase
The P24 antigen capture assay ( ELISA) which uses
anti P24 antibody as the solid phase can be used for
this.
The test is positive in about 30% of infected persons.
In the first few weeks and in the terminal phase, the
test is uniformly positive.

SANDWICH ELISA
182

Wells of microtitre plate ---coated


with specific antibody

Specimen to be tested are added

If antigen is present it binds to the


antibody

183

To detect antigen antibody


reaction antiserum conjugated
with enzyme is added

This conjugated antiserum binds


to the antigen already attatched
to the coated antibody

184

A substrate is added to know the


binding of conjugated antiserum to
antigen antibody complex

In case of binding (positive result)


enzyme acts on substrate to produce
color

Every step of ELISA test incubation


and washing is done to wash out
unbound reagents

185

USES
persons recently exposed to risk of infection and in
whom antibody test is negative.
Infants born of seropositive mothers.
screening blood donors

ANTIBODY DETECTION( SEROLOGICAL


TESTS)
186

2 TYPES
SCREENING TESTS,
SUPPLEMENTAL OR CONFIRMATORY
TESTS.

SCREENING TESTS
187

ELISA
Principle
viral antigen is coated on surface of
microwells

The test serum is added

Anti- human goat immunoglobulin


linked to a suitable enzyme is added,
followed by a colour forming
substrate

Results read by ELISA reader.

Main requirements
188

1.Serum or plasma
2.Diagnostic kit

Microwell strips coated with purified HIV-1 & HIV-2 antigens


Enzyme conjugate (peroxidise labelled purified HIV-1 and
HIV-2 antigens),
Buffered substrate: contains hydrogen peroxide in citrate
buffer, PH 5.2,
Chromogen solution: contains tetramethylbenzidine (TMB)in
dimethyl sulfoxide(DMSO),

189

stopping solution: 1.5N sulphuric acid,


washing solution,
negative control, positive control
conjugate diluents, diluents for reagents and specimens

3.Microplate ELISA reader

Procedure
190

Add 25l of diluents in each well.


Add 75l of specimen , negative control , positive
control in different wells of the microstrip and mix
well.
Cover with adhesive film and incubate.
Aspirate the contents and wash the wells.

191

Add 100l of the conjugate. Mix well and incubate


Aspirate the contents and wash the wells
Add 50l each of buffered substrate and
chromogen solution.
Incubate
Add stop solution and read absorbance

192

Interpretation:
Positive result - colour
Negative result - no colour

193

USE
Posses high sensitivity
Have a broadly reactive spectrum
Simple to perform
Can be automated for handling large sample at a time
DISADVANTAGES
Not highly specific
May give a few false positive results
It is inconvenient for testing single sample quickly.

194

FALSE POSITIVE ELISA


Presence of rheumatoid factor
Autoimmune disorders

False negative ELISA


Early infection before antibodies are detectable( 6-20%)
Some unusual HIV-1 subtypes (e.g.,subtype 0) may have
negative screening tests.

SALIVA
195

Is an acceptable alternative to serum for antibody testing by


ELISA

Salivary IgA levels to HIV decline as infected patients become


symptomatic

prognostic indicator

Orasure - commercially available

The test relies on the collection of oral mucosal transudate ( IgG


antibody ).

Advantage over serum


196

sensitivity and specificity - 95% and 100%. (Tamashiro and


Constantine, 1994; Tess et al., 1996; Emmons, 1997; Malamud,1997).

Can be collected non-invasively,

eliminates the risk of infection

Useful in special populations in whom blood drawing is difficult, i.e.


individuals with compromised venous access (e.g. injecting drug
users),patients with haemophilia
Crit Rev Oral Biol Med 13(2):197-212 (2002)

FUJIREBO AGGLUTINATION TEST


197

Ag coated gelatin particles are agglutinated by


antibody
Use simple & convenient
Disadvantage false positives

KARPAS TEST
198

Slide immuno-peroxidase reaction


HIV infected cells are fixed on teflon coated slide
well
Test serum added
Horse radish peroxidase labelled antihuman
globulin-substrate
Use simple
Disadvantage evaluation is subjective

INDIRECT IMMUNO FLUORESCENCE

Fluorochrom
e
Labeled
Anti-Ig

Unlabeled
Ab

HIV antigen is fixed on a slide.


The Pt serum is applied to the
slide.
For detection of this Ag Ab
reaction, fluorescein tagged Ab to
human globulin is added
If test is positive fluorescence
occurs at the combining site.

Ag

199

SUPPLEMENTAL TEST
200

Western blot test


Principle:
HIV proteins are separated according to their
molecular weight by polyacrylamide gel electrophoresis
gel electrophoresis --- samples are placed in wells in a
soft agar gel and subjected to an electric current
Phosphate groups in DNA--- give the entire molecule an
negative charge--- which moves towards the positive
charge in the gel

201

Rate of movement ---based on the size of the


fragment

Larger fragments move slowly--- remain nearer


the top of the gel

Smaller fragments--- migrate faster and


positioned farther from the wells

202

HIV proteins are blotted on to strips of


nitrocellulose paper

strips are reacted with test sera, with enzyme


conjugated anti-human globulin

substrate is added to produce colour bands

Main requirements
203

1) serum(or plasma)
2) Diagnostic kit:
a) Nitrocellulose strips activated by the transfer of
HIV-1 viral proteins.
b) Conjugate: Alkaline phosphatase(AP)-labelled
antihuman IgG goat antibodies.
c)
Buffered-substrate: Nitro-blue Tetrazolium
(NBT).
d) Diluent solution(wash solution).

Procedure
204

Interpretation of the results


205

Bands HIV antigen bound to


antibody

Compared with the positive control


strip containing the antibodies for
all HIV antigens

206

Test is positive if bands occur


at two locations gp 160 or gp120
and gp 24 or gp41

Test is negative- no bands are


present at any HIV antigen

Test is indeterminate- if the


bands are present ,but not at the
criteria location

207

Disadvantage
Interpretation remains subjective & demands
considerable experience
Indeterminate results

208

False negative WB results


may be due to Early infection before antibodies
are detectable.(6-20%)

Some unusual HIV-1 subtypes (eg:subtype 0)

209

False positive WB test


Hyperbilirubinemia
Presence of antibody to another human retrovirus
Human leukocyte antigen antibodies

210

Indeterminate WB test
Recent HIV infections.
Advanced immunodeficiency because of loss of
antibodies.

POLYMERASE CHAIN REACTION(PCR)

A technique by which minute


quantities of specific DNA or RNA
sequence can be enzymatically
amplified to the extent that a
sufficient quantity of material is
available for detection

GOLD STANDARD in diagnosis


of HIV in all stages.
211

212

Two types
1)PCR for DNA of provirus present in the infected
host cell.
2) RT-PCR for HIV RNA, which detects free virus
present in the plasma.

213

In the DNA PCR, peripheral lymphocytes are


lysed and the proviral DNA is amplified.

RT PCR - target is RNA


cDNA is first produced from RNA with the help of
enzyme RT and then cDNA is amplified

214

PCR uses the same events which occur naturally


in the synthesis of DNA
Opening up of double strand
Using exposed strands as templates
Addition of primers
Action of DNA polymerase

215

Initiation of reaction requires PRIMERS ---which


are synthetic oligonucleotides( short DNA ) strands--of known sequence
15-30 nucleotides bases ---- serve as landmarks of
DNA amplification
They take the place of RNA primers in the cell
Depending on the DNA being replicated the primers
can be random or highly specific

REQUIREMENTS
216

To keep DNA strands separated --- high


temperatures
Taq polymerase Thermus aquaticus( thermophilic
bacteria)
Two short primers anchor point for polymerase
and as initiator of the copying process.
All four DNA nucleotide building blocks (dATP,
dGTP, dCTP, dTTP)

PROCEDURE

3 steps
Denaturation
Annealing
Extension

217

218

Denaturation--- DNA is
separated into two
strands

Priming--- primers are


added, bind to the
complementary strand
of test DNA

219

Extension
DNA polymerase & raw
materials in the form of
nucleotides are added
Beginning at the free
ends of the primers,
polymerase extend the
molecule by adding
appropriate nucleotides

Uses:
220

PCR can detect as low as 20 copies/ ml of plasma


and is highly sensitive and specific test.
Early detection of HIV infection in new born.
Results available in 24 -48 hrs (viral culture
takes 2-4 weeks)
Detection of HIV infection during window period
To confirm indeterminate WB results

221

Limitations :
its cost
Need for sophisticated laboratory equipment
Are indicated only when other methods cannot
give a definitive results

Algorithm for AIDS tests

ELISA
antibodie
s

222

Positive
Repeat ELISA
Positive
Positive

HIV infection
Positive

confirm
with WB
indeterminate result

suspect HIV

PCR for
HIV
Negative

infection

negative

Not HIV

Non specific tests


223

To establish the immunodeficiency in HIV infection


Total leucocyte and lymphocyte count demonstrate
leucopenia and a lymphocyte count usually below
200/mm3
T cell subset assays. Absolute CD4 +T cell count will be
usually less than 200/mm3
T4:T8 ratio is reversed.
Platelet count will show thrombocytopenia
IgG and IgA levels - RAISED
Lymph node biopsy showing profound abnormalities

T CELL SUBSET ASSAYS


224

Flow cytometry
Measurements are made while cells in a liquid
suspension are forced to flow one at a time
through a measuring device.

Counting & examining cells suspended in


stream of fluid.

Method

A beam of laser light is


directed onto a stream of fluid
containing suspension of cells.
A number of detectors are
aimed at the point where the
stream passes through the light
beam.
Each cell passing through the
beam scatters the light.
Scattered light is picked up the
detectors & analysed by
computers

225

Uses
226

In AIDS patients, Lymphopenia is largely due to


progressive decrease in CD4 and increased CD8 T
lymphocytes
Normal CD4/CD8 ratio is 2:1 AIDS patients have
reversed
Antiviral therapy is recommended if CD4
count<500 / cu mm.

LABORATORY DIAGNOSIS OF VIRAL INFECTIONS


238

Specimen collection
Cultivation of viruses
Virus isolation
Microscopy
Demonstration of virus antigen
Demonstration of antibodies

SPECIMEN COLLECTION
239

Cervical, conjunctival swab, nasopharyngeal


aspirate, stool, throat, urethral swab, vesicle fluid

Collected and transported with least delay

Most viruses are heat labile so refrigeration is


essential during transport.

CULTIVATION OF VIRUSES
240

Three methods are employed for cultivation of viruses

1. Inoculation into animals


2. Embryonated eggs
3. Tissue cultures.

241

1. Animal inoculation:
Infant suckling mice are the most widely employed
animals in virology
Infant mice are used in the isolation of coxsackie and
arboviruses
Mice may be inoculated by several routes
intracerebral, subcutaneous, intraperitoneal or
intranasal.

242

After inoculation animals are observed for the signs of


diseases or death.
Later on they are sacrificed and the tissues are tested
for the presence of the virus
The viruses are identified by neutralization test using
anti viral sera
In some viruses inclusion bodies may be observed in
stained smears
Other animals such as guinea pigs, rabbits and ferrets
are used in some situations.

243

Uses
Primary isolation of certain virus
Used for study of pathogenesis of viral diseases,
To study viral oncogenesis.

EMBRYONATED EGG INOCULATION


244

Good Pasture (1931) first used embryonated hens egg for


cultivation of viruses

Embryonated hens eggs 7 to 12 days old are inoculated by


several routes--- chorioallantoic membrane, allontoic cavity,
amniotic sac and yolk sac

After inoculation eggs are incubated for 2 -9 days

245

Chorioallantoic membrane(CAM)
Mainly for growing pox virus- produces visible lesions called
(pocks).
Each infectious virus particle can form one pock.
Pock counting there fore indicates the no of viruses in the
inoculum
Different viruses produce pocks of different morphology

Inoculation into the allantoic


cavity is employed for growing
influenza virus for vaccine
production.

Inoculation into the amniotic sac


- primary isolation of the
influenza virus.

Yolk sac inoculation is used for


the cultivation of some viruses
and certain bacteria

246

TISSUE CULTURE
247

Three types of tissue cultures are available:

Organ culture - Small bits of organs can be maintained


in tissue culture growth medium for days and weeks,
preserving their original architecture and function

Organ culture are useful for the isolation of highly


specialized viruses of certain organs

E.g. tracheal ring culture for corona virus

248

Explant culture
Fragments of minced tissue can be grown as
explants embedded in plasma clots. They may
also be cultivated in suspension.
This method is now seldom employed in
virology.

CELL CULTURE
249

Routinely employed for diagnostic virology


Tissues --- dissociated into the component cells by the action of
proteolytic enzymes such as TRYPSIN
The disassociated cells are washed counted suspended in the
growth medium.
The cell suspension is distributed in glass or plastic bottles or
petridish
on incubation the cells adhere to glass or plastic surface and
divide to form a confluent monolayer sheet of cells with in one
week

250

Based on their origin, chromosomal characters


and the number of generations through which
they can be maintained

cell cultures are classified into three types:


1. primary cell culture
2. diploid cell strains
3. continuous cell lines

PRIMARY CELL CULTURE


251

These are normal cells freshly taken from the organs


of animal or human being and cultured.
They are capable of very limited growth in culture
5-10 divisions
Use
For the isolation of viruses
For vaccine production.

DIPLOID CELL STRAINS


252

These are cells of a single type that contain the same number of chromosomes as
the parent cells
They can be subcultured for a limited number

After about 50 serial subcultures they undergo senescence and the cell strain is
lost

Diploid cells are employed for the production of viral vaccines

DIPLOID cells developed from the human fibroblasts are susceptible to a number
of human viruses

Useful for isolation of fastidious pathogens

CONTINUOUS CELL LINES


253

These are cells of a single type, usually derived from


cancer cells
capable of continuous serial cultivation indefinitely
hence the name continuous cell lines
Their chromosomes are haploid
These cells grow faster
These cell lines may be maintained by serial subculture
or stored in the cold(-70c)

DETECTION OF VIRUS GROWTH IN CELL CULTURES


254

cytopathic effect:

many viruses cause morphological changes in cultured cells in which they grow

these changes can be readily observed by microscopic examination of the


cultures

these changes are known as cytopathic effects (cpe) viruses causing cpe --cytopathogenic viruses

syncytium formation

cell necrosis and lysis

cellularclumping

rounding of cells

discrete focal degeneration

255

HAEMADSORPTION

When haemagglutinating viruses grow in cell cultures, their


presence can be indicated by the addition of guinea pig
erythrocytes to the cultures.

If the viruses are multiplying in the culture, the erythrocytes will


adhere to the infected cells. This is known as 'haemadsorption'.

Specific antiserum against the virus blocks the haemadsorption

256

INTERFERENCE

The growth of a noncytopathogenic virus in cell


culture can be tested by the cytopathogenic virus

The growth of the first virus will inhibit infection by


the second virus by interference

257

TRANSFORMATION: Tumour forming viruses induce

cell transformation and loss of contact inhibition

so that growth appears in a piled-up fashion producing


micro tumors

Some herpes viruses, adenoviruses and retroviruses can


transform cells

258

IMMUNOFLUORESCENCE: viruses can be

detected in infected cells by staining with


fluorescent conjugated antiserum and
examined under the fluorescent microscope
for the presence of virus antigen.

259

Detection of enzymes the virus isolates can be


identified by detection of viral enzymes in the culture
fluid
Reverse transcriptase enzyme can be detected in
retroviruses.

COMPLEMENT FIXATION TEST


260

CFT is based on the principle of fixation of complement factors


to antigen antibody complexes which is detected by an indicator
system consisting of sheep RBC & antibodies to sheep RBC

Complement is a non specific protein present in normal human


or animal serum which are activated by Ag Ab reaction and lead
to a number of biologically significant consequences.

Complement fixation tests are most commonly used to assay for


antibody in a test sample.

METHOD

Antigen is mixed with the test serum to be


assayed for antibody
antigen -antibody complexes are allowed
to form

If no antigen antibody complexes are


present in the tube, none of the
complement will be fixed

However, if antigen antibody complexes


are present, they will fix complement and
thereby reduce the amount of complement
in the tube

261

After allowing complement


fixation by any antigen antibody
complexes, a standard amount of
red blood cells, which have been
precoated with anti-erythrocyte
antibodies is added.

If all the complement is present


all the red cells will be lysed.

262

Conclusion
263

Viruses are obligate intracelluar organisms which


do not have a cellular organization
They multipy by a complex process and
unaffected by antibacterials antibiotics
They have the ability to cause very large number
of human diseases
So they should be diagnosed, isolated &cultured
for accurate treatment

REFERENCES
264

Text book of oral pathology-shafer,Hine ,Levy-6th edition


Oral and maxillofacial pathology-nevllie,Damm,Allen,Bouquot3rd edition
Text book of Microbiology. R. Ananthanarayana. 6th edition
Medical Microbiology & Immunology.Warren Levinson 7th
edition.
Text book of immunology. Kuby
Microbiology A Systems ApproachMarjoie Kelly, Park
Dent Clin N Am 49 (2005) 1529

265

Thank
you
THANK YOU

266

Question and answers

268

Eclipse phase
From the stage of penetration of virus
into the host cell till the appearance of
first infectious virus progeny particle, the
virus cannot be demonstrated inside the
host cell
This period is known as eclipse phase

269

Other name for zoster sine herpete?


Zoster sine eruptione

270

Droplet nuclei
Droplet nuclei droplets of size less than
5m which cannot travel more than 3
feet

271

Other conditions with Warthin kinldey


giant cells?
Lymphomas , Kimuras disease, AIDS
related lymphoproliferative, lupus
erythematosus

272

ELVIS

Enzyme Linked Virus Inducible System


- new 24hrs culture test for detecting
VZV from occular samples.

273

What is HIV Disease?

HIV disease is used to broadly describe the


disease or illness caused by infection with
the human immunodeficiency virus
If untreated, the disease typically progress
slowly from asymptomatic infection to
worsening immunocompromise to AIDS
In general, this process takes about ten
years, it vary widely from person to person.

274

Vaccine against HIV infection?

Enormous effort has been devoted to the development of a


vaccine against human immunodeficiency virus (HIV)

size of the HIV genome is about ten thousand nucleotides,


this results in all daughter virions having a different genome
leading to rapid population polymorphism

Therefore a vaccine against a laboratory strain will be


unlikely to protect against the strains in the population

New strategies that can cover the vast genetic diversity of


HIV are needed for the development of a potent vaccine

275

Principle of PCR
minute quantities of specific DNA or RNA
sequence can be enzymatically amplified
to the extent that a sufficient quantity of
material is available for detection

276

Herpes barbae
Herpes simplex spread over the
bearded region of the face into the minor
injuries created by daily shaving leading
to condition known as herpes barbae

277

Other possible sites for latency of HSV-1


Nodose ganglion of vagus nerve
Dorsal root ganglion
Brain

278

Which lesions are more painful herpes


simplex or herpes zoster?
Lesions of herpes zoster are more painful

279

Significance of area of involvement in


zoster sine herpete
Severe pain, hyperasthesia over a
specific dermatome--- involvement
dermatome

280

Koplik spots
Immune reaction to the virus in the
endothelial cells of dermal capillaries
plays a role in the development of spots

281

Cell tropism

Tropism - Biological phenomenon,


indicating growth of biological organism in
response to environmental stimuli.

Cell tropism refers to the way in which


different viruses/ pathogens have evolved
to preferentially target specific host species
or specific cell types

282

TB in AIDS
Caused by Mycobacterium. Avium and M.
intracellulare , M. bovis, M.
scrofulaceum, M. africanum and M.
haemophilum usually found in
immunocompromised patients
Oral lesions are uncommon, when
present tongue is frequently
Affected areas --- chronic ulcerations or
exophytic proliferative masses

283

Confirming the diagnosis of TB is often


difficult in AIDS patient because 80% do
not react to tuberculin skin test
Identifying organisms by examining AFB
stained specimens and confirming its
presence on culture are important
Management is difficult --- increasing
drug resistance

284

Southern blot test


DNA fragments are first separated by
electrophoresis and then denaturated and
transferred to a special filter
DNA probe is then incubated with the sample
Whenever this probe encounters the
segment for which it is complementary it will
attach and form a hybrid
Development of the hybridization pattern will
show up as one or more bands

285

This method is a sensitive and specific


way to isolate fragments from a complex
mixture
To find specific gene sequences on DNA

TRIDOT
286

A circular specimen port A; and a large


elliptical reaction port B.
Antigens derived from HIV -1 and HIV-2
are immobilized in two separate spots on
the reaction zone of part-B.
A third spot serves as the internal control
for monitoring the performance of the test.

Principle:
287

A diluted specimen is introduced to port


A. Anti HIV antibodies bind specially to
the HIV antigens and migrate along the
chromatographic strip.
In the reaction zone of part B, the antigen
antibody complexes bind to immobilized
HIV antigens in a dual recognition step.
The resultant complex is reacted with a
streptavidine- alkaline phosphatase
conjugate.

288

Virus culture is not used routinely for


diagnostic purpose?
Serial subcultures ---- undergo
senescence and the cell strain is lost

289

Why antibodies are preferred to antigen


for screening?

Cost effective
Antibodies directed against a specific
antigen can be produced invitro by
hybridoma technique