1
Q
What are viruses?
A
Submicroscopic pathogens whose size is measured in nanometers
2
Q
Describe the basic structure of a virus
A
- A core of DNA or RNA packaged into a protein coat ( capsid)
- in some viruses, the capsid is surrounded by an outer envelope of glycolipids and proteins derived from the host-cell membrane
- obligate, intracellular
3
Q
What are the steps of the basic virus lifecycle?
A
① attachment of the virus to a receptor on the host cell surface
② penetration of virus into host cell through endocytosis
③ degradation of the viral capsid and subsequent release of viral nucleic acid.
④ transcription to produce additional viral nucleic acid
⑤ translation of viral nuclei acid to produce viral proteins
⑥ assembly of the viral components to produce intact virions
⑦ budding off the host-cell membrane or host-cell lysis results
⑧ release of viral progeny
4
Q
What are the defenses types against a virus?
A
- Innate defenses
- humoral antibody responses
- cell-mediated immunity
5
Q
Describe innate defenses against viruses
A
- First line of protection (initial barriers)
- skin and mucous membrane barriers
- recognition of PAMPs on virus-infected host cells
- interferons alpha and beta
6
Q
What occurs if the initial barrier of innate defense does not work?
A
Other innate defenses are activated when cells of the innate immunity recognize PAMPs on surface or within virus infected host cells
7
Q
What are other innate defenses against viruses?
A
- viral cells are stimulated to produce IFN-alpha and IFN- beta after recognizing viral RNA by TLRs
- IFNs inhibit viral replication by inducing transcription of several genes that code for proteins with antiviral activity
- they also enhance activity of NK cells
8
Q
How do NK cells fight against viruses?
A
- Bind to virus-infected cells and release proteins such as a perforin and granzymes, causing cells to die and release viruses and are now accessible to antibody molecules
9
Q
What plays a key role in preventing the spread of viral infection through neutralization?
A
Antibodies
10
Q
Describe antibodies role in preventing the spread of viral infection through neutralization
A
-Involves production of antibodies that are specific for a component of the virus that binds to a receptor on the host-cell membrane
- when these neutralizing antibodies bind to the virus, they prevent it from attaching to and penetrating the host cell. IgA plays large role in this.
- IgG and IgM can bind to viruses in blood stream and inhibit dissemination of infection
- IgG and IgM activate complement
- IgG also promote phagocytosis of viruses and promote destruction of viruses through ADCC
- IgM also viral particles by agglutinating them
11
Q
What does intracellular viruses require?
A
Cell-mediated immunity
12
Q
What cells have key roles in cell mediated immunity?
A
-Th1 cells
- cytotoxic T cells
13
Q
Describe Th1 cells actions that occur in cell mediated immunity
A
- Produce IFN-gamma, which induces an antiviral ‘ state within the virus-infected cells
- produce IL-2, which assistsin development of effector cytotoxic T cells
14
Q
Describe cytotoxic T cells actions that occur in cell mediated immunity
A
- CD8 + cytotoxic T cells become programmed to expand in number and attack the virus infected cells
- CD8 is a co-receptor of t-cell receptor on cytotoxic T cells that must bind to viral antigen complexed with MHC class I on infected cell surfaces
15
Q
What occurs after cytotoxic T cells bind to viral antigen complexed with MHC class I?
A
- Stimutate granules in the cytotoxic T cells to release perforin and granzymes entering the pores
16
Q
What are perforins?
A
- protein that produces pores in the membrane of the infected host cell
17
Q
What are granzymes?
A
- Protease that enter cells through pores created by perforin.
18
Q
What occurs once granzymes enter the viral infected cell?
A
- Activate apoptosis in the host cell, interrupting the viral-replication cycle and resulting in release of assembled infectious virions
- The free virions can then be bound by antibodies
19
Q
Describe humoral antibody responses
A
- Antibodies attack free virus particles
- viral neutralization, opsonization, C’ fixation, and ADCC
20
Q
What are viral escape mechanisms? And examples?
A
① mutations results in production of new viral antigens (influenza viruses undergo frequent genetic changes)
② viruses block action of immune system components (HSV can bind C3b)
③ suppression of the immune response (CMV reduces MHC I
④ immune function altered (EBV stimulates polyclonal B-cell activation)
⑤ latent state is established (VZV remains latent in nerve cells)
21
Q
What laboratory test are ran to detect a viral infection?
A
- Serological tests
-distinguish between current and past infection
-antibody titers used to monitor course of infection
-assess immune status - molecular
-detect active infection
-quantitative tests→ guide antiviral therapy
22
Q
How are current and past infections detected?
A
- IgM (+) and IgG (+/-) → current or recent infection. (congenital)
- IgM (-) and IgG (+) → past infection
23
Q
What does the presence of virus-specific IgG indicate?
A
Immunity to virus
24
Q
What is the hepatitis virus?
A
- Hepatitis is the inflammation of the liver
25
What is hepatitis caused by?
- radiation '
- chemical toxins
- secondly to other disease
- cirrhosis (alcoholism)
- drugs
- hypothermia
- bacteria
- fungi
- parasites
26
What types of hepatitis are transmitted by fecal-oral route?
Hep A (HAV)
Hep E (HEV)
27
What types of hepatitis are transmitted through parenteral route?
Hep B (HBV)
Hep D (HDV)
Hep C (HCV)
28
What are indicators of Hepatitis ?
- flu-like symptoms early on
- pain in upper right quadrant of abdomen
- hepatomegaly and liver tenderness with profession
- jaundice
- dark urine
- light feces
- elevated bilimbin and liver enzymes (ALT)
29
Describe hepatitis A
- nonenveloped, single strand RNA virus
- belongs to hepatovinis genus of picornarviridaefamily
- acute hepatitis in majority of adults
- infections in children are usually asymptomatic
- formalin-activated vaccine
- HAV immune globulin may be recommended for unimmunized persons exposed to virus
30
How is hepatitis A transmitted?
- Fecal-oral route
- close person-to-person
- ingestion of contaminated food or water
31
What is the treatment for hepatitis A?
-supportive → bedrest, nutritional support, and medication for fever, nausea, and diarrhea
32
What is critical for establishing a diagnosis of hepatitis A?
- Serological tests antibody
33
what is acute infectionS or immunity to HAV indicated by?
(+) IgM anti-HAV → acute infection
(+) total anti- HAV along with (-) IgM anti-HAV
34
What sheds from the feces of someone with hepatitis A?
HAV antigens
35
What are hepatitis A antibodies must commonly detected by?
EIAs
Chemiluminescent microparticle immunoassays
36
When is IgM anti-HAV detectable in a patient with hepatitis A?
Onset of clinical symptoms and declines to undetectable levels by 6 months
37
Describe tests for total HAV antibodies
- Detect IgM but predominantly IgG, which persists for life
38
Although IgM anti-HAV is primary marker to detect acute hepatitis. What do you have to look out for?
False-negative results due to early phase of infection
39
What is the most common format of these molecular methods of HAV?
-RT-PCR
- used to test samples of food or water suspected of transmitting virus
40
Describe hepatitis E
- nonenveloped, single stranded RNA virus with four genotypes
- HEV-1 and HEV-2 are transmitted primarily through ingestion of faces- contaminated drinking water
- HEV-3 and HEV-4 are transmitted mainly by consumption of infected pork
-most asymptomatic or self-limiting infections
- can detect HEV RNA in blood or stool during acute infection
-belong to hepevirus, in the family hepeviridae
41
What indicates an acute infection of Hep E?
IgM anti-HEV
42
What indicates past exposures of Hep E?
IgG anti-HEV
43
What does diagnosis of HEV rely on?
- Serology to detect antibodies to the virus (EIAs)
- molecular methods to HEV nucicic acids
44
What is used to detect later stages, past exposures, and identify seroprevalence of the infection in Hep E?
Immunoassays for IgG anti-HEV
45
What is the gold standard for diagnosis of acute HEV infections?
qPCR, quantization of HEV nucleic acid
46
What testing can be done for immunocompromised patients that are suspected of having Hep E?
- molecular testing for HEV RNA (qPCR)
- LAMP
47
When does HEV RNA become undetectable in blood?
3 weeks after symptoms onset
48
When does RNA HEV become undetectable in stools?
5 weeks after onset of systems
49
How long is the incubation period for Hep A?
Average of 28 days
50
Describe hepatitis B
- DNA virus with 8 genotypes (A-H)
- belongs to hepadnaviridae family
- acute infection → symptoms increase with age
- chronic infection → 6 months or more, occurs in 90% of infected infants and 10% of infected adults
- preventable with immunization
- hepatitis B immune globulin (HBIG) may be given to unimmunized persons exposed to HBV
51
What does chronic hepatitis B increase the risk of?
Liver cirrhosis
Hepatocellular carcinoma
52
Describe hepatitis B surface antigen (HBsAg)
- First HBV marker to appear (2-10 weeks after exposure
- protein on outer envelope of virus
- excess circulates in virus-like particles in blood
- marker for active HBV infection
- component of hepatitis B vaccine
53
Describe hepatitis B antigen (HBeg)
- Protein in core of HBV
- marker of active viral replication
- indicates high degree of infectivity
54
What are serological markers of HBV antigens?
HBsAg
HBeg
55
What are the antibodies markers for HBV?
Anti-HBc
Anti-HBe
Anti-HBs
56
Describe anti-HBc of HBV
- Directed against hep B core antigen
- IgM anti-HBc indicates current/recent infection
- “core window”
-total anti-HBc consists mainly of IgG and can indicate a current or past - infection
57
What is core window?
- Period when neither HBsAg nor HBsAb can be detected in the serum of the patient only the anti-HBc
58
Describe anti-HBe of HBV
- Directed against HBeAg
-indicates recovery from hepatitis B
59
Describe anti-HBs of HBV
- Directed against HBsAg
- indicates immunity to hepatitis B.
60
How is HBV transmitted?
Transmitted through parenteral or perinatal routes:
- sexual contact
- IV drug use
- during birth process
61
What is the incubation period of HBV
30-180 days
62
Describe recovery of adults with HBV
- most recovery within 6 months
- 1% develop fulminant liver disease with hepatic necrosis
63
Describe chronic hepatitis B
- Majority of infants, 10% of adults, one-third children
- most likely in immunocompromised patients
- results in liver inflammation and damage
- can be treated with anti-viral drugs
64
Describe structure of the HBV
- Nucleocapsid core surrounded by outer envelope of lipoprotein
- core of virus contains circular partially double- stranded DNA
65
How are serological markers of HBV used?
- Differential diagnosis of HBV
- monitoring course of infection
- assessing immunity to virus
- screening blood productsfor infectivity
66
Why is HBsAg important marker of HBV?
- Indicator of active infection
- monitoring course of infection and progression
- screening of donor blood
67
How are serological markers for hepatitis B most commonly detected? Describe
- Commercial immunoassays (EIAs and CLIA) ''
- typically automated
- excellent specificity and sensitivity
- false-negatives and false-positives occur
- positive results should be verified by repeat testing followed by confirmation with additional assay ( HBsAg neutralization test or molecular methods that detect HBV DNA
68
What is the method of choice to quantify HBV DNA
qPCR
69
What are molecular methods used to detect HBV DNA?
- PCR
- qPCR
- branched DNA signal amplification
70
What is considered a successful treatment for HBV?
1- log10 reduction in HBV DNA levels
71
Describe hepatitis D
- RNA virus that requires presence of HBV with 3 genotypes
- superinfection of chronic HBV carriers → chronic liver disease with accelerated progression to cirrhosis and liver failure
- co-infection with HBV → usually results in acute, self-limited hepatitis
72
In HDV, what is the marker for active viral replication?
HDV RNA
73
What do co-infection of HDV look like?
Positive for anti-HDV and IgM anti-HBc
74
What do chronic cases of HDV results look like?
Positive for anti-HDV and IgG anti-HBc
75
How is HDV transmitted?
Through parents evil or perinatal routes
76
What marker appearance would make cause to test for HDV?
-HBsAg
- involves detection of HDV antibodies and HDV RNA
77
What testing is used to distinguish HBV and HDV from an acute infection or superinfection?
Serology testing
78
What does the presence of IgG anti-HDV indicate?
- Acute, chronic or past infection
79
What ave molecular methods used to detect HDV RNA?
- RT-PCR assays
- also provides quantitative results that can be use to monitor the response of patients to antiviral therapy
80
Describe hepatitis C
- enveloped, single-strandedRNA virus with 7 genotypes
- belongs to flaviviridae family and genus hepacivirus
- most infections are asymptomatic at first but develop into chronic liver disease
- genotyping is used to determine best therapy
81
How is HCV transmitted?
- Exposure to contaminated blood, sexual contact, and perinatal exposure
82
What is detection of anti-HCV IgG used for?
- Screening and diagnosis of HCV
83
What is qualitative HCV RNA used for?
Confirmation
84
What are quantitative molecular tests used for?
Monitor viral load during antiviral therapy
85
What is the most common blood born infection in the United States?
HCV
86
What is the HCV genotype that is most prominent
In the United States?
Genotype 1
87
Why is it difficult to create a vaccine for HCV?
- The variability of HCV
- its ability to undergo rapid mutations within its hosts
88
What is the incubation period of HCV?
2 weeks - 6 months (7 weeks average)
89
What does chronic HCV lead to?
- Cirrhosis of liver ''
- increased visit of hepatocellular- carcinoma
- rheumatoid conditions
- glomerulonephritis
- vasculitis
- neuropathy
- dermatological systems
90
What is the standard treatment for HCV?
- A combination of pegylated IFN-alpha and ribavirin
- very effective in genotype 2 and 3
- only effective for 50% of genotypes
91
What is anti-HCV IgG most commonly detected by?
- EIAs and CLIAs that use recombinant and synthetic antigens
- rapid immunoblot is alternative
92
When do antibodies become detectable in HCV?
8-10 weeks after exposure and remain positive for a life time
93
What could cause a false positive result in serological testing of HCV?
- Because of cross-reactivity in persons with other viral infections or autoimmune disorders
94
What test is recommended for HCV RNA confirmation?
- Nucleic acid testing
95
What do qualititative tests distinguish between in HCV?
- Presence or absence of HCV RNA
96
What are qualitative molecular assays used for in HCV?
- To confirm infection in HCV antibody positive patients
- detect infection in antibody negative patients ho are suspected of having HCV
- screen blood and organ donors for HCV
- detect perinatal infections in babies
97
What are types of quantitative molecular assays for HCV
- RT-PCR
- qPCR
- bDNA application
98
What are quantitative molecular assays for HCV used for?
- Monitor amount of HCV RNA, or viral load, carried by patients before, during and after antiviral therapy in chronic patients
99
What is the goal of antiviral therapy of HCV?
When patient continuously tests negative for HCV RNA 12 or 24 weeks after therapy is completed
100
Describe genotyping of HCV
- To determine exact genotype and subtype of vines responsible for infection
- ran on all HCV positive patient before antiviral therapy
- important to identify genotype because they vary in their responses to different antiviral drugs
101
What can genotyping of HCV be performed by?
- PCR amplification and sequencing of target gene
- PCR followed by identification of the target gene with genotype-specific probes
- qPCR
102
What is the reference method of genotyping for HCV? Why?
- PCR/direct sequencing (Sanger sequencing)
- because it provides precise information abort genomic variability of the virus during course of infection
103
Describe herpes vinises
- Large, complex DNA viruses that are surrounded by a protein capsid, an amorphous tegument, and an outer envelope
- herpesviridae family → includes 8 viruses
104
Describe epstein-barr virus (EBV)
-DNA herpes virus most commonly transmitted by intimate contact with salivary secretions
- begins in oropharyux in B cells and epithelial cells and spreads through lymphoreticular system
105
What are diseases caused by Epstein-Barr virus?
Infectious mono
Lymphoproliferative disorders
Certain Malignancies ( burkitt lymphoma)
106
How can epstein-barr virus be transmitted?
- Salivary secretions ( most frequent)
- blood transfusions
- bone marrow
- solid- organ transplant
- sexual contact
- perinatal exposure
107
What cells are infected in the oropharynx in a patient with EBV?
Epithelial cells
B cells
108
How does EBV enter the B cells?
By binding to surface CD21
109
What are early antigens (EAs) of EBV?
- antigen produced during the initial stages of viral replication in the lyric cycle
- can be classified into two groups (based on location): EA-D and EA-R
110
Describe EA-D
In EBV, Early antigens that have a diffuse distribution in nucleus and cytoplasm
111
Describe EA-R
In EBV, early antigens restricted to cytoplasm only
112
Describe latent antigens of EBV
- Appear during the period of the lyric cycle following viral DNA synthesis
- includes the viral capsid antigens in the protein capsid and the membrane capsid in viral envelope
113
What antigens appear during the latent stage of EBV?
- EBV nuclear antigen (EBNA) proteins
- EBNA-1
- EBNA-2
- EBNA-3
- EBNA-4
- EBNA-5
- EBNA-6
- LMP-1
- LMP-2A
- LMP-2B
114
What are classic symptoms of IM?
- Fever
- lymphadenopathy
- sore throat
- fatigue
- lasts 2-4 weeks
115
What are laboratory findings of IM?
- Absolute lymphocytosis
- 20% or more atypical lymphocytes
- heterophile antibodies→ IgM antibodies
116
Describe treatment of IM
Mainly directed at alleviating symptoms
117
What are heterophile antibodies?
- Antibodies that are capable of reacting with similar antigens from two or more unrelated species
118
What procedures can be performed for detection of IM heterophil antibodies?
- Monospot (rapid slide agglutination)
- paul-bunnell test
- rapid agglutination tests
- immunochromatographicassays using purified bovine RBC extract as antigen
119
When can false positives occur in IM testing?
- Lymphoma
- viral hepatitis
- malaria
- autoimmune disease
- errors in interpretation
120
What can AID in diagnosis of IM?
-Testing for EBV-specific antibodies
- helpful is patients that test negative for heterophil antibodies or determine past exposure.
- detected by IFAs, blot techniques, ELISA, CLIA or flow cytometric microbead immunoassays
121
What is the gold standard of EBV serology methods?
- IFAs
- labs prefer to use EIAs or CLIA tests because the are automated and easier to interpret
122
What is the most useful marker for acute IM?
IgM antibody to the VCA
123
What results typically indicate primary IM infection?
-Presence of lgM anti-VCA and anti-EA-D
- absence of anti-EBNA
124
What type of tests are best to detect EBV in immunocompromised patients:
Molecular tests
125
How is cytomegalovirus transmitted?
- DNA herpes virus is transmitted through oral secretions, genital secretions, congenitally, or by transfusion/ transplantation
126
Describe cytomegalovirus (CMV)
- Healthy individuals may be asymptomatic or develop a mononucleosis- like syndrome
-in immunocompromised persons, CMV can disseminate to lungs, liver, GI tract, CNS, and eyes and cause life-threatening infections
- may cause congenital defects and decreased survival in infants
127
What are types of testing for CMV?
- Direct virus detection
- serology
128
Describe direct virus detection of CMV
- viral culture
-sample placed with a cell type that the virus being tested for can infect
-if the cells show changes, known as cytopathic effects, then the culture is positive
- ID of CMV antigens
- molecular tests for CMV DNA
129
Describe serology methods for CMV
- used to screen blood and organ donors; pregnant women
- presence of IgG anti-CMV indicates infection
- low avidity antibodies indicate recent infection
- most beneficial in determining past exposures
- usually semi- or fully automated EIAs
130
What is the most common cause of congenital infections
CMV
131
What indicates CMV?
- Characteristic cytopatic effects (CPEs) that produce enlarged, rounded, refractile cells
132
Describe the CMV antigenemia assay
- Uses immunocytochemical or immunofluorescent staining to detect the CMV lower-matrix protein pp65 in infected leukocytes from peripheral blood or cerebral spinal fluid
- 2-4 hours to perform
133
What is the most widely used molecular test for CMV? Why?
- RT-PCR
- sensitive, simple to perform and provide quantitative results
134
Describe VZV
- DNA herpes virus
- cause of: varicella (chickenpox) and herpes zoster ( shingles)
- preventable by vaccine
135
How is VZV transmitted?
- By inhalation of infected respiratory secretions or aerosols from skin lesions
136
Describe varicella
- Highly contagious illness
- blister-like rash
- intense itching
- fever
137
What are the activities of VZV in the primary infection?
- Thought to travel from the skin lesions and the blood to sensory neurons, where it deposits its DNA and establishes a lifelong latent state in the dorsal root, autonomic and cranial ganglia
138
Describe herpes zoster
- Painful vesicular rash caused by the vines moves down the sensory nerve to the dermatome supplied by that nerve
- most common complication being postherpetic neuralgia
-life threatening complications: herpes ophthalmicus which leads to blindness, pneumonia, and visceral involvement
139
What is the definitive diagnosis of VZV?
- identifying VZV (or one of its products) in sick lesions, vesicular fluids or tissue
- used for atypical cases
140
What is the method of choice to detect VZV DNA?
qPCR
141
How is serology testing useful with VZV?
- Determines immunity to VZV in health-care workers, pregnant women, and organ transplant candidates
142
What cells can be detected in VZV patients?
- Tzanck cells
143
When is quantitative PCR useful with VZV?
- Monitoring response of immunocompromised patients to antiviral drugs
144
What type of specimens are PCRs van on for VZV?
- Scab
- Skin swabs
- vesicular fluid
- throat swabs
- cerebrospinal fluid
- blood
- saliva
-tissues
145
Why is testing for VZV IgM not done?
① IgM antibodies to VZV may not be detectable until the convalescent stage of illness
② they cannot distinguish between primary and reactivated infection
③ may not be free of IgG antibodies when semi's processed for testing
146
Describe serology testing for VZV
- most of them detect total VZV antibody, which consists primarily of IgG
- most reliable and sensitive method → FAMA (reference method)
-most commonly used → ELISA
147
Describe FAMA
- Fluorescent antibody to membrane antigen
- detects antibody to the envelope glycoproteins of the virus
- requires live, virus-infected cells
- not suitable for large scale testing
148
Why can a false positive be caused by using ELISA to detect VZV?
- The method can detect low-levels of antibodies that do not confer long-term protection to varicella
149
Describe rubella virus
- Enveloped, single-stranded RNA virus
- Cause of German measles
- can be prevented with immunization
- genus rubivirus and belongs to family Togaviridae
- 12-23 day incubation period
- after incubation, virus replicates in upper respiratory tract and cervical lymph nodes, then travels through blood stream
150
How is the rubella virus transmitted?
- Respiratory droplets
- across the placenta
151
What can rubella virus cause?
- deafness
- eye defects
- cardiac abnormalties
- mental retardation
- motor disabilities
- miscarriage
- stillbirth in infants born to infected mothers
152
What are symptoms of rubella virus?
- Erythematous (macropapular rash). Appears first on the face, then spreads to the trunk and extremities
- low grade fever
- malaise
- swollen glands
- upper respiratory infection lasting 1-5 days
- 50% infected with Rubella are asymptomatic
- arthritis
153
What population has severe consequence with the rubella virus? Why ?
- Pregnant women, especially in first trimester
- may cause miscarriage, stillbirth, or congenital rubella syndrome
154
What can occur if infants are born with congenital rubella syndrome (CRS)
- Many abnormalties
-Deafness
-eye defects (cataracts and glaucoma)
-cardiac abnormalties
-mental retardation
-liver and spleen damage
-motor disabilities
155
What type of methods are ran for rubella virus?
- Serology
- viral culture
- molecular methods
156
Describe serology testing of rubella virus
- Presence of IgG is used to screen for immunity
- congenital infection is indicated by rubella- specific IgM or fourfold rise in IgG
- low avidity antibodies indicate recent infection
157
Describe viral culture of the rubella virus?
- Diagnosis can be obtained this way
- viral growth is slow
- may not produce characteristic CPE
158
If CPE is absent in the viral culture of rubella, what should be done next?
Viral nucleic acid can be identified by RT-PCR or viral proteins can be detected with IFA or EIA
159
What is the most widely used test for rubella virus?
- Molecular methods → RT-PCR
- used to detect rubella RNA
160
What clinical samples can be tested with RT-PCR for rubella?
- Chorionic villi
- placenta
- amniotic fluid
- fetal blood
- lens tissue
- products of conception
- pharyngeal swab
- spinal fluid
- brain tissue
161
What are the most common methods used to confirm Rubella?
- Serology tests (rapid and cost effective)
- detect rubella antibodies
- ELISA → most widely used
- hemagglutination inhibition (HI)
- latex agglutination
- immunoassays
162
Describe ELISA testing of rubella
- Specific solid phase capture ELISAs can be used to detect IgM rubella antibodies
163
What assays can simultaneously detect measles, mumps, rubella, and varicella?
- Automated chemiluminescence assays
- multiplex bead immunoassay
164
How is a primary rubella infection indicated?
- Presence of rubella-specific IgM antibodies
OR
- four-fold or greater rise in rubella specific IgG antibody titers collected at least 10-14 days apart
165
What are the causes for false-negative and false positives in rubella?
False negative → if sample is obtained too early
False-positive → individuals with parvovirus infections, enterovirusinfections, heterophil antibodies or rheumatoid factor
166
Describe test ran to confirm positive IgM results of rubella
- EIA → measures avidity of rubella IgG antibodies, which helps distinguish between recent and past infections
- low antibody avidity → recent infection
-high antibody avidity→ past infections
167
Describe Laboratory testing of congenital rubella infection
- Begins with serological evaluation of the ' mothers antibodies and measurement of rubella-specific IgM antibodies in fetal blood, cord blood, or neonatal seruml
- any positive IgM results should be confirmed by viral culture, RT-PCR amplification, or demonstration of persistentLY nigh taters of rubella IgG antibodies after 3- 6 months of age
168
Describe rubeola
- RNA virus single-stranded RNA virus
- genus morbillivirus in the paramyxoviridae family '
- after initial infection of the epithelial cells in upper respiratory tract,rubeola virus is disseminated through the blood to multiple sitesin body, such as the skin, lymph nodes and liver
- preventable by immunization
- diagnosis is usually based on clinical presentation and confirmed by serology
169
How is rubeola transmitted?
Through respiratory droplets
170
What does rubeola cause?
- Measles
- subacute sclerosing panencephalitis (SSPE)
171
What is the incubation period for rubeola?
10-12 days
172
What are the symptoms of rubeola?
- Fever
- cover
- runny nose
- conjunctivitis
- Koplik spots appear on mucous membrane of inner cheeks or lips
173
What is a the rubeolla virus characterized by?
Erythematous (maculopopular eruption) that begins at hairline, then spreadsto the face and neck and gradually moves down to trunk, arms, hands, legs, and feet
174
What complications can measles result in?
- Diarrhea
- otitis media
- croup
- bronchitis
- pneumonia
- encephalitis
175
What is SSPE associated with ?
Extremely nigh titers of rubeola antibodies
176
What is the preferred method to detecting IgM antibodies in rubeola?
- IgM capture ELISA method
- 3-4 days after symptoms
177
What is the preferred method of detect IgG antibodies of rubeola?
- ELISA
- detectable 7-10 days after symptoms and persist for life
- IgG antibodies indicate immunity
178
When are molecular methods used to detect rubeola RNA?
- In which serological tests are inconclusive or inconsistent
- can be used to genotype the virus
179
What is the preferred molecular method for detecting rubeola?
- RT-PCR
180
Describe mumps virus
- Single stranded RNA vines
- paramyxoviridae family, genus ribulavirus
- most common clinical manifestation is parotitis
- replicates initially in nasopharynx and regional lymph nodes
- preventable by immunization
- diagnosis is usually based on clinical presentation
- confirmation is done by culture or RT-PCR
181
How is mumps transmitted?
Respiratory droplets, saliva, fomites
182
What are fomites?
Inanimate objects or substances that can transmit infectious organisms
183
What is the incubation period of the mumps virus?
14-18 days
184
How does the mumps virus function?
- Virus spreads from blood to various tissues:
-meningesof brain
-salivary glands
-pancreas
-testes
-ovaries
- produces inflammation at site
185
What is parotitis?
- Inflammation of the parotid glands
- occurs in 30.40% of patients with mumps
186
What is the gold standard for detecting the mumps virus
- Culture
187
What are the preferred specimens for a culture of mumps?
Buccal swab
Saliva from buccal cavity
188
What is the primary diagnostic test for mumps? Why?
- RT-PCR
- because it is more sensitive than serology
189
Describe serological testing of mumps
- Provides simple means of confirming mumps ' ' diagnosis but has some important limitations
- EIAs and IFA mostly only measure IgG mumps virus antibodies
- most commonly used → ELISA
- use of solid-phase IgM capture assays reduces incidence of false positivesbecause of rheumatoid factor C
190
What is a current or recent infection of mumps indicated by?
- Presence of mumps-specific IgM antibody in a single serum sample or by at least a four-fold rise in specific IgG antibody between two specimens collected during the acute and convalescent phases of illness
191
Describe human t-cell lymphoytropic virus type I and type II (HTLV-I & HTLV-2)
- Closely related retroviruses that preferentially infect T-lymphocates ( usually CD4+)
- serological tests for antibodies to HTLV-I/II are used to diagnose infections and screen blood donors.
- have RNA as their nucleic acid and enzyme reverse transcriptase
- was 3 structural genes: gag, pol, and env and region called pX
-ELISA or CLIA are used to screen
-western blot or LIA are used for confirmation of positive results
192
What does HTLV-I cause?
-adult T-cell leukemia/lymphoma (ATLL) and HAM/TSP
193
How are HTLV types I and II transmitted?
- Mainly blood borne
- sexual contact
- perinatal (especially breastfeeding)
194
What is "gag?”
Codes for viral core proteins
195
What is “pol?"
Codes for viral enzymes
196
What is "env?"
Encodes proteins in viral envelope
197
What is “pX?”
Encodes several regulatory proteins, including tax and Rex
198
What is reverse transcriptase function of HTLV?
To transcribe the viral RNA into DNA
199
What are the 4 subtypes of ATLL? What are they characterized by?
- acute
- T-cell non-Hodskin's lymphoma
- chronic
- smoldering
- a monoclonal proliferation of matureT cells that express the surface markers CD3, CD4, and CD25
200
How is HAM/TSP characterized?
- Slowly progressive weakness and stillnessof legs
- back pain
- urinary incontinence
201
What has been associated with HTLV-I?
- variety of autoimmune and inflammatory disorders
-uveitis (intraocular inflammationof the eyes)
-infective dermatitis
-myositis (inflammation of the muscles)
-arthropathy (inflammation of the joints)
202
When do HTLV antibodies develop?
30-90 days after exposureto the virus and persists for life
203
When is a sample considered positive for HTLV-I performed with western blot?
-if visible bands are produced for one of the env proteins (either gp46 or gp62/68) AND one of the gag proteins (either p19, p24,or p53
204
Describe PCR methods for HTLV testing
- detects HTLV type I and II DNA
- Can be used to monitor the proviral load in patients during therapy
205
Describe toxoplasmosis
- Found in feces of house cats and rodents (other mammals)
206
How is toxoplasmosis transmitted?
- Accidental ingestion of oocyst (fecal contamination of meat, raw milk, etc)
- transplacental transmission
207
How does on individual prevent congenital toxoplasmosis?
-avoid touching mucous membranes of mouth and eyes while handling raw meat
-wash hands and Kitchen surfacesafter coming in contact with raw meat
-not allow flies and cockroaches access to food
-avoid contact (or wear gloves) when handling cat litter boxes or gardening
208
What are signs and symptoms of toxoplasmosis?
- Usually asymptomatic, organism can multiply in any organ of body
- similar to mono (if symptoms are mono)
- spontaneous recovery
209
Describe congenitalinfections of toxoplasmosis
- Of most concern '
- result in CNS malformation and mortality of the neonate
- 75% have no symptoms at birth, disease is dormant, discovered only when neurological problems such as blindness occurs
210
What is the method of choice for a laboratory diagnosis for toxoplasmosis?
EIA
211
Describe TORCH testing
- Consists of tests for antibodies to four organisms that cause congenital infections transmitted from mother to fetus
T → toxoplasmosis
O→ others
R→ rubella
C→ CMV
H→ herpes simplex virus (HSV)
212
What are the " others " of TORCH?
- Syphilis
- HBV
- coxsackie virus
- EBV
- VZV
- human parvovirus
213
What viruses affect the respiratory system?
- Adenovirus
- influenza
- mumps
- measles
- RSV
214
What virus affects the skin?
Arbovirus
215
What virus effects the GI tract?
Rotovirus
216
What is the role of CTLs in immune responses against viruses?
Destroy virus-infected host cells
217
A newborn suspected of having a congenital viral infection should be tested for virus - species antibodies of which class?
IgM
218
The serum of an individual who received all doses of the hepatitis B vaccines should contain what?
Anti-HBs
219
Heterophile antibodies are routinely detected by their reaction with what?
Bovine erythrocyte antigens
220
What is the method of choice for detecting VZV infection inimmunocompromised hosts?
(Real-time) qPCR
221
Describe human immunodeficiency virus (HIV)
- Causes AIDS (acquired immunodeficiency syndrome)
- two types: HIV-1 and HIV-2
222
Describe HIV-1 (will be referred to as in this set)
- Cause of most HIV infections worldwide
- 4 groups → M, O, N, P
- 9 subtypes in group M→ A, B , C, D, F, G, H, J, K
- group M is responsible for most cases
- predominant subtype→ C
- most prevalent in US→ B
- belongs to genus lentivirinae of virus family Retroviridae
- decrease in cell population is a hallmark feature
223
Describe HIV-2
- Originated in west Africa
- causes fever cases
- less pathogenic
- lower transmission rate
224
What are ways HIV can be transmitted?
- Sexual contact involving exchange of body fluids → responsible for majority of cases
- contact with blood or other body fluids
- perinatal → before, during and after( breast milk)
225
What body fluids are infective with HIV?
- Blood
- semen and vaginal secretions
- synovial, pleural, peritoneal and pericardial
- CSF
- breast milk.
226
What are characteristics of HIV
- Retrovirus
- contains two copies of ssRNA
- reverse transcriptase transcribes the viral RNA into DNA
- surrounded by a protein coat (capsid)
- outer envelope of glycoproteins embedded in a lipid bilayer
227
What is gp120
Docking glycoprotein
228
What is gp41?
Transmembrane glycoprotein
229
What is p17?
Matrix protein
230
What is p24?
Capsid
231
Why is HIV considered a retrovirus?
because it contains RNA as its nucleic acid and a unique enzyme called reverse transcriptase
232
Describe the HIV structure
- spherical particle
- includes 3 structural genes: gag, pol and env
- 100-120 nm in diameter
- contain inner core with two copies of single stranded RNA surrounded by protein coat (capsid) and an outer envelope of glycoproteins embedded in lipid bilayer
- glycoproteins are knob-like structures that are involved in binding the virus to host cells during infection
233
describe gag of HIV
- codes for p55
- p55 is a precursor for p6, p9, p17 and, p24
- all are located in the nucleocapsid of the virus
- p17 embedded in the internal portion of the envelope (matrix)
- p6, p9, and p24 located in capsid that surrounds the internal nucleic acids
234
Describe env of HIV
- codes for the glycoproteins gp160, gp120, and gp41 which are all found in the viral envelope
- gp160 is a precursor protein that is cleaved into gp120 and gp41
-gp120 forms protruding knob structures on the outer envelope
- gp41 is a transmembrane glycoprotein that spans the inner and outer membrane and attaches to gp120
-both gp120 and gp41 are involved in fusion and attachment of HIV to receptors on the host cells.
235
Describe pol of HIV
- codes for enzymes necessary for HIV application
- located in the core of the virus in association with HIV RNA
- enzymes: reverse transcriptase (p51), RNAse H (p66), integrase (p31) and protease (p10)
236
describe p66 of pol in HIV
enzyme involved in the degradation of the original HIV RNA
237
describe p31 of pol in HIV
an enzyme that mediates the integration of viral DNA into the genome of infected host cells.
238
Describe p10 of pol in HIV
cleaves precursor proteins into smaller active units used to make the mature virions
239
Briefly Describe replication of HIV
- attachment of HIV to host cell
- main target: CD4 Th
- coreceptor required
240
What are the steps of HIV replication?
1. Binding - HIV binds to receptors on surface of CD4 cell
2. Fusion - HIV envelope and and CD4 cell membrane fuse and allows virus to enter cell
3. Reverse transcription - inside cell, HIV uses and releases reverse transcriptase, which HIV RNA into HIV DNA. This allows HIV to enter nucleus of CD4 cell
4. Integration - inside nucleus, HIV releases integrase. Uses integrase to insert its viral DNA into the DNA of the CD4 cell
5. Replication - HIV uses machinery of CD4 cell to make long chains of HIV proteins. These proteins are building blocks for more HIV
6. Assembly- new HIV proteins and HIV RNA move to surface of cell and assemble into immature HIV
7. Budding - immature HIV pushes out of host CD4 cell surface. New HIV release proteases, which break down the long protein chains in immature HIV, forming mature HIV
241
what is T-tropic or X4 strains of HIV?
that preferentially infect T-cells
242
what is M-tropic or R5 strains of HIV?
can infect T-cells and macrophages
243
what chemokine receptor is required for HIV to enter T-lymphocytes?
CXCR4
244
what chemokine receptor is required for HIV to enter macrophages?
CCR5
245
when does HIV not virally replicate?
during the latent stage
246
what are the type of immune responses for HIV?
- innate
- humoral antibody production
- cell-mediated immunity
247
Describe the innate defenses against HIV
- NK cells medicate cytolysis of HIV infected cells
- dendritic cells stimulate release of cytokines that have antiviral effects
248
Describe humoral antibody production against HIV
- Antibodies are detected by 6 weeks after infection.
- Antibodies produced later may prevent HIV from infecting host cells and participate in ADCC
249
Describe cell-mediated immunity against HIV
- T cells produce cytokines with antiviral activity
- CTLs destroy HIV-infected host cells
250
what are the HIV escape mechanisms?
1. genetic mutaions rapidly occur, generating new viral mutants with altered antigens
2. down regulates expression of MHC-I molecules on infected host cells
3. can survive as a patent provirus for prolonged periods
4. As a result, HIV persists and destroys the immune system
5. CD4 T cells are the prime targets of destruction, resulting in reduced effectiveness of antibody and cell-mediated immune responses
251
How is HIV suspected to kill or render function of CD4 Th cells?
1. loss of plasma-membrane integrity because of viral budding
2. destruction by HIV-specific CTL
3. viral induction of apoptosis
252
what is the central role in the immune system for CD4 T cells?
regulating the activities of B and T cells
253
what is the acute stage of HIV characterized by?
- rapid bursts of viral replication before the development of HIV-specific immune responses
- high levels of viremia is found in this stage
- decrease in CD4 T-cell number
254
what are the symptoms of acute stage of HIV?1
- flu-like symptoms
- IM-like symptoms
- many are asymptomatic during this stage
255
describe HIV latent stage
- decrease in viremia
- increase in CD4 T-cell number
- symptoms are subtle or absent
256
describe the last stage of HIV
- full blown AIDS
- characterized by profound immunosuppression with very low numbers of CD4 T cells
- resurgence of viremia
- patients demonstrate neurological symptoms
- appearance of life-threatening opportunistic infections and malignancies
257
what are the symptoms for infants with AIDS?
- failure to thrive
- persistent oral candidiasis
- hepatosplenomegaly
- lymphadenopathy
- recurrent diarrhea
- recurrent bacterial infections
258
Describe antiretroviral therapy (ART)
- drugs that block various steps of the HIV replication cycle
-nucleoside analog reverse transcriptase inhibitors
-nonnucleoside reverse transcriptase inhibitors
-protease inhibitors
-integrase inhibitors
-fusion inhibitors
-CCR5 antagonists
-Post attachment inhibitors
- are most effective when used in combination
- has significantly improved morbidity and mortality of HIV-infected persons and has reduced the rate of perinatal transmission
259
what are ways to prevent HIV?
- screening blood/ organ donors for HIV
- education of the general public on HIV transmission, safety measures
- precautions for health-care workers
- vaccine-being researched
260
Describe screening and diagnosis of HIV
- previous algorithm and test methods
- current algorithm and test methods
261
Describe testing of HIV
- performed on infants
262
Describe previous testing algorithm of HIV
- screen for HIV-1/HIV-2 antibodies by ELISA or rapid EIA
- confirm positive test results by repeating ELISA, followed by Western Blot
263
Describe the Western Blot test
- also known as immunoblotting
- tests for a specific protein with a protein mixture
- is performed after gel-electrophoresis has separated protein
- uses antibodies to identify specific proteins
- separated proteins are transferred onto nitrocellulose or nylon membranes and identified by specific antibodies that are tagged by a secondary protein
264
Describe the wester blot interpretations of HIV
- no band: negative
- positives are harder to interpret
- CDC says must have antibody against two or three of the following bands:
-p24
-gp41
-gp120/160 (these bands are very close together and is hard to distinguish between the two.
265
Describe the current CDC testing algorithm for HIV
- initial screening: HIV-1/2 antigen/antibody combination immunoassay
- If positive: run HIV-1/2 differentiation immunoassay
-HIV-1(+) & HIV-2 (-): HIV-1 antibodies present
-HIV-1(-) & HIV-2(+): HIV-2 antibodies present
-HIV-1(+) & HIV-2(+): HIV-1 and HIV-2 antibodies present
- If both HIV negative, run HIV-1 NAT
-HIV-1 NAT (+): acute HIV-1 infection
266
what is the principle of the fourth generation HIV-1antibody/HIV-2 antibody/p24 antigen combination immunoassay?
1. incubate patient serum with solid phases onto which HIV-1antigens, HIV-2 antigens and antibody to HIV-1 p24 have been bound
2. Following incubation, HIV-1 or HIV-2 antibodies in the patient sample will bind to their respective antigens.
3. HIV-1 p24 antigen in the patient sample will bind to anti-p24 solid phase
4. Wash, then add conjugate consisting of labeled anti-24 labeled HIV-1/HIV-2 antigens
5. Following, incubation, wash, and addition of trigger solutions or substrate/stop solution, relative light units or optical absorbance are measured
6. Confirm positive results with rapid EIA
267
What are some ways the HIV is monitored?
- peripheral blood CD4 T-cell counts
- Quantitative viral load assays
- Drug resistance and tropism
268
Why do false-positives occur in the 4th generation HIV-1/2 antibodies and p24 antigen test?
- heat inactivation
- repeated freezing and thawing
- presence of heterophil antibodies
- passive immunoglobulin administration
- administration of of an experimental HIV vaccine to patient
269
What is seroconversion?
the change of a serological test result from antibody negative to antibody positive.
270
What are the two markers used to monitor HIV for disease progression and guide the treatments?
- the peripheral blood CD4 T-cell count (best indicator of immune functions)
- HIV-1 RNA level (viral load)
271
Describe CD4 T-cell enumeration
- CD4 T-cell numbers are the best indicator of immune function in HIV-infected individuals
- incubate peripheral blood with fluorescent-labeled anti-CD4; analyze results by flow cytometry
- in untreated patients, CD4 T-cell number declines progressively, and CD4 T: CD8 T-cell ratio is less than 1:1
- CD4 T-cell count of less than 200/ul indicates stage 3 HIV
- A significant decline in CD4 T-cell count over time may indicate a need to change ART or administer prophylactic therapy for certain infections
272
what is the peripheral blood CD4 T-cell count in a healthy individual?
450-1500 cells/ul
273
what is the gold standard for enumerating CD4 T cells?
immunophenotyping with data analysis by flow cytometry
274
Describe the CDC classification list of HIV
0-5
- 0: early infection of patient who tested positive for HIV in initial screening but has a negative or indeterminate confirmatory test
- 1-3: based on peripheral blood CD4 T-cell count or percentage. If this information is missing, classified as unknown
275
Describe quantitative viral load assays
- measure amount of HIV RNA circulating in patient plasma
- methods: qPCR and bDNA
- HIV RN detectable about 11 days after infection
- successful therapy with ART results in the decline in the viral load to an undetectable antigen
- Patients with persistently increased viral load should undergo drug resistance testing and may need a possible change in ART
276
Why are viral load tests used?
- help predict disease progression
- monitor patient response to ART
- guide treatment plan
277
Describe the viral load assays of HIV RNA
-based on amplification methods that increase the number of HIV RNA copies
-most common: PCR and bDNA assay
278
what are the 2 PCR methods that have been developed to detect HIV nucleic acid?
- RT-PCR (not usually used)
- qPCR
279
what is the basic principle of RT-PCR for HIV RNA?
- to amplify a DNA sequence that is complementary to a portion of the HIV RNA genome
280
What are disadvantages of RT-PCR for HIV RNA?
- limited dynamic range
- highly susceptible to cross-contamination with extraneous nucleic acid
281
what is the basic principle of bDNA assay
based on amplifying the detection signal generated in the reaction.
- accomplished by using a solid-phase sandwich hybridization assay that incorporates multiple sets of oligonucleotide probes and hybridization steps that produce a series a branched molecules.
282
what are disadvantages of bDNA?
- requires larger sample volume
- lacks internal controls
- lower specificity
283
what are drug resistance testing methods for HIV?
- genotype resistance assays
- phenotype resistance assays
- Tropism testing
284
Describe genotype resistance assays of HIV
- performed in clinical laboratory settings
- HIV reverse transcriptase and protease genes from RNA in patient plasma are amplified by RT-PCR
- products are sequenced and analyzed with software for mutations
- Results are reported as: Resistance, Possible resistance, no evidence of resistance
285
Describe phenotype resistance assays of HIV
- Determine ability of HIV from clinical samples to grow in the presence of antiretroviral drugs
- involve sophisticated technologies only performed by specialized reference laboratories.
286
-Describe tropism testing of HIV
- genotypic or phenotypic assays are performed to determine if the patient has virus that will bind to the CCR5 co-receptor and be responsive to CCR5 antagonists
287
Describe testing of infants younger than 18 months in HIV
- all pregnant women should be tested for HIV
- Maternal antibodies in infant serum can complicate serological test results
- molecular methods are used for diagnosis
- Qualitative HIV-1 DNA PCR using infants peripheral blood mononuclear cells is the preferred method
- Serological testing at 12-18 months of age may be used to confirm the diagnosis
288
HIV visions bind to host T cells through which receptors?
CD4
CCR5
289
What is typical of the latent stage of HIV infection?
- Proviral DNA is attached to cellular DNA
290
What is the drug zidovudine an example of?
Nucleoside analogue reverse-transcriptase inhibitor
291
Why would a false negative result in a test for HIV ?
Collection of the test sample before seroconversion
292
What does the conjugate used in 4th- generation immunoassays for HIV consists of labeled?
- HIV-1 and HIV-2- specific antigens plus antibody to p24
293
What is the first detectable antibodyin serum after infection with hepatitis B?
HBc
294
What is the difference between HAV and HBV incubation periods?
- HAV→ short
- HBV→ long
295
How do you interpret these results?
Anti-HAV (IgG)→ negative
Anti-HAV (IgM)→ negative
HBsAg→negative
Anti-HBc→ positive
Anti-HBs→ negative
HBeAg→ negative
- HBV in cove window phase
296
What is the most common cause of congenital infections?
CMV
297
What populations does VZV cause severe complications in?
- Immunocompromised patients
- pregnant women
298
What is the clinical importance of CMV?
Life threatening for immunocompromised patients and infants
299
A patient has IgG and IgM antibodies against viral capsid antigens of EBV. What does this patient have
- Current EBV infection
300
What are heterophile antibodies routinely detected bytheir reactionwith this agent?
Bovine RBC antigens
301
What does the presence of IgM anti-rubella antibodies in serumfrom an infantborn with a rash?
Congenital infection with rubella virus
302
What are the a most commonly used tests to detect mumps virus,?
RT-PCR
ELISA
303
What is method of choice for defecting VZV in immunocompromised hosts?
RT-PCR
304
Describe Tzack smear
Scrap lesion with scalpel but put on slide and stain, then look for giant multinucleated cells
305
What does p24 encode for?
Gag
306
How should these results be interpreted HIV?
ELISA: positive
Repeat ELISA: neg
Western blot: no bands
Negative for HIV
307
What type of cells can HIV infect?
- primarily lymphocytes
- monocytes
308
What does the HIV cove consists of?
Two identical strands of RNA
309
What is uncaring of genome, making RNA into DNA?
Reverse transcriptase
310
Why is HIV called a retrovirus?
Can transcribe RNA into DNA
311
A 22-year-old male sees his physician for an annual checkup. He reportedhaving flu-like symptoms including fever, sore throat, and lymphadenopathy several months ago.he has lust about 10 pounds but otherwisefeels fine. HIV testing is recommended and ordered
ELISA→ positive
Western blot→ positive
CD4+ T-cell count is 500/ml
What disease and stage is this ?
HIV positive in latency stage 1
MLT immunology
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