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INFLUENZA

History
o The word Influenza comes from the Italian language meaninginfluence and refers to the cause of
disease; initially, this ascribed illness to unfavorable astrological influences.
o The symptoms of human influenza were clearly described by Hippocrates roughly 2,400 years ago.
o The first influenza virus to be isolated was from poultry, when in 1901 the agent causing a diseases
called fowl plague was passed through Chamberland filters, which have pores that are too small
for bacteria to pass through.

Clinical Features
o Causative agent: Haemophilus influenzae
o Single stranded, segmented RNA, enveloped virus
o Contain several structural proteins that include the matrix protein, nucleocapsid protein, and three
proteins that function in RNA replication and transcription.
o Three types: A (cause more extensive epidemics and disease) B, and C.
o The most striking characteristic: presence of multiple copies of two glycoproteins that project like
spikes from the membrane.
a. Hemagglutinin (HA)
- Required for entry into the cell.
- Agglutinate RBCs.
- Initiation of viral attachments, penetration and fusion to the host plasma cell membrane.
b. Neuraminidase (NA)
- Facilitates release of virus from the cell.
- Enzyme that catalyzes the hydrolysis of linkages that join terminal sialic acid residues to specific
sugar molecules (D-galactose or D-galactosamine).
- Allow the virus to penetrate the protective mucus layer and gain access to underlying
epithelial cell in respiratory tract.
- Prevent self aggregation of newly formed viruses.
- Serve as the basis for a commercially available test kit designed to detect viral antigens in
clinical specimen.

Minor mutations in the HA and NA that occur frequently are referred to as antigenic
drift.
Major antigenic change occurs that obviates the advantage of pre-existing immunity
is referred to as antigenic shift.

o The influenza virus infects the respiratory epithelial cells.


o Mode of Transmission: Respiratory secretions
o Incubation Period: 24-96 hours; 48 hours (most common)
o Recovery: 1-2months
o Symptoms:
Fever
Headache
Photophobia
Sore muscles
Malaise
Sore throat
o Secondary bacterial pneumonia may occur late in the disease. Reyes syndrome has been
associated with influenza. Salicylates (Aspirin) is used during the initial phase of infection.
o RIDTs can be useful to identify influenza virus infection as a cause of respiratory outbreaks in any
setting, but especially in institutions (i.e., nursing homes, chronica care facilities, and hospitals), cruise
ships, summer camps, schools, etc. Positive RIDT results from one or more ill persons with suspected
influenza can support decisions to promptyl implement infection prevention and control measures for
influenza outbreaks.

Immunology
o Role of T cells and NK Cells
- CD8+ T cells and NK Cells induce osmotic lysis of virally infected cells by secreting perforins (a
molecule that inserts into the infected cell membrane.
- CD4+ T cells play an important role in viral infections in that they are required for differentiation
of nave CD8+ T cells to mature cytotoxic cells.
o Role of Antigen Presenting Cells
- APCs endocytose the influenza virus, with or without opsonins, when it is outside the cell.

Immunization
o Nasal Administration of Live Attenuated Vaccines
- A trivalent, live, attenuated influenza vaccine (CAIV-T) consisting of two influenza A strains and
one influenza B strains has been used to test an intranasal delivery system.
- Serve as an alternative vaccines that use injections of of inactivated virus for protection
against influenza in children.
o Role of Adjuvants in Vaccines
- Chrion has recently introduced the influenza vaccine, Fload.
- Fload is a recombinant protein subunit vaccine that incorporates the MF59 adjuvant instead
of alum (commonly used adjuvant)
- MF59 consists of a submicron oil-in-water emulsion (5% v/v squalene, 0.5% v/v Span 85 and
0.5% v/v Tween 80).
Laboratory Diagnosis
1. Endogenous Viral Enzyme Assay (EVEA)
- The ZstatFLU test (ZymeTx) is an endogenous viral encoded enzyme assay (EVEA) that
indirectly determines the presence of influenza A and B.
- Specimen: Throat swab
- Detects viral neuraminidase activity
- Substrate: modified form of N-acetylneuramic acid linked to a chromogen.
- Colored medications or residual blood may result in an off white color.
o Positive: Chromogen is released as blue precipitate.
- A positive result means that the RIDT detected influenza viral antigen, but does not
necessarily mean variable influenza virus is present or that the patient is contagious.
o Negative: The spot on the collection membrane device is white, or any shade other than blue.
- A negative result means that the RIDT did not detect any influenza viral antigen.
2. Optical Immunoassay
- OIA using silicon assay surface technology (SILAS) has been developed for the rapid
detection of influenza A (or B) viral antigen rom clinical specimens.
- Specimens: sputum, throat swab, nasal aspirate.
- Two major steps:
i. Capture of Influenza A and B nucleoproteins.
ii. Detection of that protein
o Positive: Surface will appear as a purple-blue color.
- The positive predictive value of an RIDT (the proportion of patients with positive results who
have influenza) is highest when influenza activity is high in the population being tested (e.g.
community).
- The negative predictive value of an RIDT (the proportion of patients with negative results
who do not have influenza) is highest when influenza activity is low in the population being
tested (e.g. community).
3. Serological tests can indicate infection by a fourfold rise in antibody titer when direct virus detection
is not successful ( HI, EIA, microneutralization)
o Positive: A positive result is most likely a true positive result if the respiratory specimen was
collected close to illness onset (within 4 days) during periods of high influenza activity (e.g.
winter) in the population being tested (e.g. community).
o Negative: A negative is most likely a true negative result if the respiratory specimen was
collected close to illness onset ( within 4 days) during periods of low influenza activity (e.g.
summer) in the population being tested (e.g. summer) in the population being tested (e.g.
community).
4. RT-PCR can be used to monitor genetic and antigenic characteristics of circulating strains
o Positive: A positive result in a person who recently received intranasal administration of live
attenuated influenza virus vaccine (LAIV) may indicate detection of vaccine virus. LAIV contains
influenza virus strains that undergo viral replication in the respiratory tissues of lower temperature
(e.g., nasal passages) than internal body temperature. Since the nasal passages are infected
with live influenza virus vaccine strains during LAIV administration, sampling the nasal passages
within a few days after LAIV vaccination can yield positive influenza testing results. It may be
possible to detect LAIV vaccine strains up to 7 days after vaccination, and in rare situations, for
longer periods.
o Negative: The negative predictive value of an RIDT (the proportion of patients with negative
results who do not have influenza) is lowest when influenza activity is high in the population
being tested (e.g. community).
***False-negative results are more likely to occur when influenza prevalence is the high in the
community.
5. Fluorescent antibody staining by direct IFA, rapid immunoassays to detect influenza antigens.
o Positive: The positive predictive value of an RIDT (the proportion of patients with positive results
who have influenza) is lowest when influenza activity is low in the population being tested (e.g.,
community).
o Negative: Sensitivities of RIDTs are generally approximately 50-70%, but a range of 10-80% has
been reports compared to viral culture or RT-PCR. Specificities of RIDTs are approximately 90-95%
(range 85-100%). Thus false negative results occur more commonly than false positive results.
6. RIDTs can be useful to identify influenza virus infection as a cause of respiratory outbreaks in any
setting, but especially in institutions (i.e., nursing homes, chronica care facilities, and hospitals), cruise
ships, summer camps, schools, etc. Positive RIDT results from one or more ill persons with suspected
influenza can support decisions to promptyl implement infection prevention and control measures
for influenza outbreaks.
o Positive: False positive results are more likely to occur when influenza prevalence in the
population tested (e.g. community) is low, which is generally at the beginning and end of the
influenza season and during the summer.
o Negative: Negative results of RIDTs do not exclude virus infection and influenza should still be
considered in a patient if clinical suspicion is high based upon history, signs, symptoms and
clinical examination.

HERPES VIRUS

Clinical Features
o Herpes simplex virus (HSV) can be cultured from the oropharynx in about 1% of healthy adults and
from the genital tract of slightly less than 1% of asymptomatic adult women who are not pregnant.
o Incubation period: 2 to 12 days.
o The most frequent manifestation of HSV infection is the common cold sore or fever blister.
o Recurrent HSV disease usually results from the reactivation of latent virus resting in paraspinal or
cranial nerve ganglia that innervate the site of primary infection.
o Two cross-reacting antigen types of HSV have been identified:
a) HSV-1
- Found in and around the oral cavity and in skin lesions that occur above the waist.
- MOT: hand-to-mouth and kissing
- Most common clinical manifestation of primary HSV-1 is acute gingivostomatitis
b) HSV-2
- Isolated primarily to the genital tract and skin lesions below the waist.
- Most common cause of genital vesicular lesions in humans.
o Manifestation has been described since ancient times, it was only in 1919 that the virus was isolated
by inoculation of Herpes labialis vesicle fluid into a rabbit cornea.
o The herpes viruses are large, complex DNA viruses that are surrounded by a protein capsid, an
amorphous tegument, and an outer envelope. These viruses are all capable of establishing a latent
infection with lifelong persistence in the host.
o All the human herpesviruses are large, enveloped DNA viruses that replicate within the cells
nucleus. The virus gains an envelope when the virus buds through the nuclear membrane, which has
been altered to contain specific viral proteins.
o Herpesviridaefamily
1. Herpes simplex viruses (HSV-1 and HSV-2).
2. Varicella-Zoster (also known as human herpes virus-3 or HHV-3)
3. Epstein-Barr virus (HHV-4)
4. Cytomegalovirus (HHV-5)
5. Human Herpes Viruses 6, 7, and (HHV-6, HHV-7)
6. Kaposis sarcoma (HHV-8)
o The herpesviruses cause a number of clinical diseases, although they share the basic characteristic
of being cell associated, which may partly account for their ability to produce subclinical infections
that can be reactivated under appropriate stimuli.

Laboratory Diagnosis
o Methods for the laboratory diagnosis of HSV include isolation of the virus and direct detection of
antigen in tissues or cytologic preparation through the use of immunofluorescence or
immunoenzyme methods. In addition, detection of the virus in body fluids (using monoclonal
antibodies) can be performed with immunoassays or immunoblot techniques.
o For identifying immunoglobulin class of antibodies produced in response to the virus may be
determined using Indirect Immunofluorescence and Enzyme Immunoassay Method.

VARICELLA-ZOSTER VIRUS
Clinical manifestation
o Member of Herpesviridae family
o Virion is comprised of an icosahedral capsid (contains DNA genome)
o Primary infection with the virus results in the clinical manifestations of chickenpox.
o Reactivation of the virus results in the characteristic clinical manifestation of (zoster), known as
shingles.
Epidemiology and Etiology
o Varicella primarily affects children age 2 to 5 years.
o The presumed route of transmission is through the respiratory tract.
o Zoster
- Shingles
o Varicella
- Varicella has an incubation period of 14 to 17 days.
- There may be a 1- to 3-day prodromal period of fever, headache, and malaise.
- Chicken pox
Signs and Symptoms
o Complications of VZV include pneumonitis, encephalitic conditions, nephritis, hepatitis,
myocarditis, arthritis, and Reyes syndrome.
o Zoster Infection
- Zoster infection is characterized by vesicular eruptions, typically con ned to one or two
adjacent dermatomes.
- Characterized by neuralgia for a few days to weeks
o Neonatal Varicella Infection
- Neonatal varicella may be acquired in utero or in the perinatal period and can result in
congenital abnormalities.
- The infant is at greatest risk if the mothers illness occurs 4 days or less before delivery.
- Febrile purpura (few days after onset of rash)
- Postinfection purpura (1 or 2 weeks after appearance of rash)

Laboratory Diagnosis
o Serologic methods include indirect immunofluorescence, which detects antibodies to specific
membrane antigens, and EIA.
o Rapid preliminary diagnosis can also be made by direct immunofluorescence to detect viral
antigens in vesicular lesions.
o Stains reveal multinucleated giant cells called Tzanck cell
Hematoxylin-eosin
Wright-Giemsa
Toluidine blue
Papanicolau
o The best way to confirm VZV infection is to recover the virus in human diploid fibroblast cell
cultures.
o Polymerase Chain Reaction
- Most accurate and sensitive method
- Detect VZV DNA
- Specimen: CSF
o Antibodies to varicella are detectable within several days of the onset of rash and peak at 2 to 3
weeks.
o In addition, ELISA methods are valuable for assessing the immune status of adults.
o Fluorescent Antibody to Membrane Antigen (FAMA)
- most sensitive and reliable method of detecting VZV antibody
Interpretation of results
o A positive IgG result coupled with a positive IgM result suggests recent infection with varicella-
zoster virus (VZV). This result should not be used alone to diagnose VZV infection and should be
interpreted in the context of clinical presentation.
o A positive IgG result couples with a negative IgM result indicates previous vaccination to or
infection with VZV. These individuals are considered to have protective immunity to reaction.
o A negative IgG result coupled with a negative IgM result indicates the absence of prior exposure
to VZV and nonimmunity. However, a negative result does not rule out a VZV infection. The
specimen may have been drawn before the appearance of detectable antibodies. Negative
results in suspected early VZV infections should be followed by testing a new serum specimen in
2 to 3 weeks.

Vaccines
o 1995, a vaccine (Varivax) consisting a strain of live, attenuated varicella virus was licensed in US for
use in children aged 12 months or older, adolescents and adults.
o 2005, a vaccine was licensed for used in healthy children that combines the varicella vaccine with
that for measles, mumps and rubella.
o In addition, a single-agent VZV vaccine was licensed in 2006 for prevention of herpes zoster in
persons aged 60 or older, presumably by boosting the immune response.

CYTOMEGALOVIRUS

Etiology
o Human CMV
- is classified as the member of herpesviridae family (herpes viruses).
- Consists of a double stranded DNA and icosahedral capsid.
- spreads to the lymphoid tissues and proceeds to circulate to systemic lymph nodes.
- the virus finally rest in the epithelial cells of many tissues.
o During infection, CMV can be detected in three forms:
1. Mature infectious virion
2. Non infectious particles lacking a genome
3. Dense bodies
History
o 1904 first descriptive report of histologic changes characteristic of those now associated with CMV
infection was originally published.
o 1956 and 1957 CMV was isolated in the laboratory
o 1966 actual isolation of virus after transfusion and observation of elevated antibody titers occurred.
o 1881 Inclusion bearing cells were first shown by Ribbert.
o 1921 Good Pasture and Talbert were the first to suggest that the cytomegalia could be due to a
viral agent.
o 1950 Smith and Vellios showed that infection may occur in utero.

Antigens and Antibodies in CMV Infection


o Immediate Early Antigens appear within 1 hour of cellular infection before replication of viral DNA
takes place.
o Early Antigens appears 24 hours
o Late Antigens demonstrated in the nucleus and cytoplasm of infected cells at about 72 hours after
infection, or at the end of the viral replication cycle.

Epidemiology
o Transmission
- Transmission of CMV may be by the oral, respiratory, or venereal route.
- The virus has been isolated in urine, saliva, feces, breast milk, blood, cervical secretions, virus-
infected grafts from a donor, semen, vaginal fluid, and respiratory droplets.
- It may also be transmitted by the transfusion of fresh blood.
- CMV is the most common virus transmitted to the fetus.
o Latent Infection
- Persistent infections characterized by periods of reactivation are frequently termed latent
infections.
- True viral latency is defined by the presence of the genetic information in an unexpressed
state in the host cell.
o Congenital Infection
- Primary and recurrent maternal CMV infection can be transmitted in utero.

Signs and Symptoms


o Acquired Infection
- Acquired CMV infection is usually asymptomatic.
- Incubation period: 3 to 12 weeks
- Symptoms include a sore throat and fever, swollen glands, chills, profound malaise, and
myalgia.
- The following three types of CMV infections are possible in blood transfusion recipients:
1. Primary infection occurs when a previously unexposed (seronegative) recipient is
transfused with blood from an actively or latently infected donor.
2. Reactivated infection can occur when a seropositive recipient is transfused with blood
from a CMV antibodypositive or negative donor.
3. Reinfection can occur by a CMV strain in the donors blood that differs from the strain
that originally infected the recipient.
o Congenital Infection
- The classic congenital CMV syndrome is manifested by a high incidence of neurologic
symptoms, as well as neuromuscular disorders, jaundice, hepatomegaly, and splenomegaly.
- Petechia is the most common clinical sign, seen in about 50% of CMV-infected infants.

Immunologic Manifestation
o CytoGam an intravenous solution of gamma globulins, enriched for anti-CMV antibodies.
o Serologic Marker
- Before replication of viral DNA takes place, immediate-early antigens and early antigens are
present in the nuclei of infected cells.
- Immediate-early antigens appear within 1 hour of cellular infection and early antigens are
present within 24 hours.
- At about 72 hours after infection, or the end of the viral replication cycle, late antigens are
demonstrable in the nucleus and cytoplasm of infected cells.

Immunization Progress
o Adjuvants are substance added to the vaccine preparation to enhance the immune response so
that a higher antibody titer is achieved.
o MF59 preparation was shown to be more effective in clinical trials.

Laboratory Tests
o New CMV infections can be identified by testing for immunoglobulin G (IgG) antibodies on blood
samples taken at different times.
o A newer method, called IgG avidity testing, which measures antibody maturity, has been shown to
detect recent primary CMV infection reliably.
o The preferred method for diagnosis is culture of virus and/or polymerase chain reaction (PCR).
o The presence of IgG antibody in infants complicates the interpretation of serologic results during the
first 6 months of life because the antibody may be maternal in origin.
o Passive Latex Agglutination for Detection of Antibodies to Cytomegalovirus
- The CMVscan Card Test is a passive latex agglutination test for the detection of IgM and IgG
CMV antibodies.
- Screening test
- This assay can be performed qualitatively on undiluted serum to identify antibodies to CMV
and quantitatively using serial twofold dilutions to determine the titer of CMV antibody.
- Positive: If antibody to CMV is present, the agglutinated particles will be macroscopically
visible.
- Negative: The latex particles will not agglutinate in the reaction mixture and the particles will
appear smooth and evenly dispersed.
o Quantitative Determination of IgG Antibodies to Cytomegalovirus
- Diluted samples are incubated in antigen-coated wells.
- Positive: Antibodies are immobilized in the wells.
- In the presence of the enzyme, the substrate is converted to a yellow end product, which is
read photometrically for an absorbance maximum at 405 nm.
o CMV rapid culture
- Test Method: Cell culture, immunofluoresence
- Recommended use: Rapid diagnosis of CMV infection and Gold Standard test for tissue.
o CMV by Polymerase Chain Reaction
- Test Method: Qualitative PCR
- Recommended use: rapid test for diagnosing CMV in immunocompromised patients or solid
organ donors.
o CMV PCR
- Test Method: Quantitative PCR
- Recommended use: diagnose CMV infection and monitor disease state in solid organ
transplant and HIV patients.
o CMV antibodies: IgM and IgG
- Test Method: Latex agglutination
- Recommended use: Screen pregnant women and infants possibly infected with CMV.
Infants may test positive during first 6 months due to maternal antibodies. Discriminate
between current (IgM) and prior infections (IgG).
o CMV antibodies: total
- Test Method: Solid-phase agglutination
- Recommended use: screen organ donors
o CMV by Immunochemistry
- Test Method: Immunochemistry
- Recommended Use: Histologic diagnosis of CMV based on tissue from affected site.