FINAL: DETECTION & ID OF MICROORGANISMS II Flashcards
TRADITIONAL DIAGNOSTIC METHODS FOR VIRUS
- antibodies against the virus (INDIRECT)
- presence or absence of viral antigens
- growth of a virus in a culture system
- Produced the host for immune response
- Retrospective indication for infection
- Paired sera
o 1 from Acute phase
o 1 from Recovery Phase - 4x rise from acute phase
o Acute stage of virus
ANTIBODIES
best evidence for the presence of virus
IgM
- Igm may be low detection limit
- px is infected and infectious
WINDOW PERIOD
- Produced/expressed by the pathogen/virus
ANTIGENS
DETECTION OF ANTIGENS
EIA/DIRECT IMMUNOFLUORESCENT ASSAYS
- Tissue/cell culture
o Monolayer of cells
o Virus is inoculated to the culture
o Observation of CYTOPATHIC EFFECT microscopically
Growth of viruses
Culture is Gold standard for
Adenovirus, enteroviruses, CMV, influenza, and HSV
Disadvantages OF CULTURE:
o Time-consuming
o Some viruses do not present cytopathic effect
o Must be collected on acute phase
Genomes of Viruses
- Detection via:
- Detection via:
o Target amplification
PCR
RT-PCR
qPCR
TMA
o Signal amplification
bDNA
hybrid capture
- RNA VIRUS; Has Reverse Transcriptase
- Rapidly mutates, creates multiple subtypes//clades
Human Immunodeficiency virus (HIV)
Human Immunodeficiency virus (HIV) TYPE:
- More pathogenic
Type 1/ HIV-1
Human Immunodeficiency virus (HIV) TYPE:
- Minor isolate
Type 2/HIV-2
SUBTYPES OF HIV:
- 95% around the world
- Further divided into 8 clades
- A, B, C, D, E, F, G, H, J
GROUP M (MAJOR)
SUBTYPES OF HIV:
- West Africa
GROUP O (OUTLIER)
SUBTYPES OF HIV:
- Cameroon
GROUP N (NON - M & NON - O)
SUBTYPES OF HIV:
- RAREST, Cameroon
GROUP P
_________ & _________ of HIV interact with CD4- fusion
gp120 & gp41
How important are helper T cells?
They help to coordinate the immune response by communicating with and activating other immune cells
Structural genes:
- 1 envelope
- Attachment and fusion to cell
- Gp160
o Gp120
o Gp41
Env
Structural genes:
- Group Antigen gene
- Found in nucleocapsid
- p55
o P15, p17, p24
o 1st antibody to emerge in HIV
Gag
Structural genes:
- Polymerase gene
- Reverse Transcriptase
o RNA to DNA
- Integrase
o Inserts viral DNA to host DNA
Pol
HIV DIAGNOSIS:
- for antibodies produced
EIA = ENZYME IMMUNOASSAY
HIV DIAGNOSIS:
standard screening
ELISA = ENZYME - LINKED IMMUNOSORBENT ASSAY
HIV DIAGNOSIS:
- standard confirmatory test
Western Blot
HIV DIAGNOSIS:
- is applied after HIV diagnosis
- for determination of viral loads
Nucleic acid amplification
CDC criteria for western blot
2 out of 3 bands to p24, gp41 and gp120/160
Nucleic acid amplification:
- goal of Antiretroviral therapy
50 copies/ml of blood
Nucleic acid amplification:
- reduced AIDS development
10,000 copies/ml
- To know when to start ART
- Monitor efficacy of treatment
RT-PCR
Herpes virus has how many members?
25 members
Herpes simplex virus
- 2 main types
Cold sores and fever
blisters
HSV-1
Herpes simplex virus
- 2 main types:
Genital herpes
HSV-2
HSV DIAGNOSIS
- EARLY METHODS: CULTURE,ANTIGEN & ANTIBODY
- WESTERN BLOT TEST
- qPCR
- Can reactivate and found in saliva of infected persons
Epstein-Barr virus (Kissing Disease)
Epstein-Barr virus (Kissing Disease) DIAGNOSIS?
- Immunohistochemistry (IHC)
o EBV-encoded RNA (EBER) - Antibodies for EBV
- EBV nuclear antigen (EBNA)
- Southern Blot
- qPCR
- Downey cells in blood smears
- May also reactivate
- typically ASYMPTOMATIC, but can cause complications in people w/ weakened immune system
-qPCR: CMV polymerase (UL54) or
glycoprotein B (gB) gene
CYTOMEGALOVIRUS (CMV)
- Varicella Chicken pox
- Zoster - Shingles (reactivated)
- ELISA and PCR w/ hybridization/ gel electrophoresis
Varicella Zoster /Herpes Zoster virus
Varicella Zoster /Herpes Zoster virus SPECIMEN SOURCES?
- LESION SWAB, BLOOD, CSF, TISSUE BIOPSY, THROAT SWAB
- ssRNA virus,
Flaviviridae - viral hepatitis & cirrhosis, associated w/ causing hepatocellular carcinoma
- Parenteral transmission
- serology: detection of antibodies (specify western blot)
- RT - PCR, TMA, bDNA
HEPATITIS C VIRUS (HCV)
- dsDNA virus, Oncogenic
- Five genotype groups (α, β, y, μ, and v)
- detection: hybridization, amplification (PCR & hybrid capture), direct sequencing methods
Human Papilloma Virus (HPV)
HPV Five genotype groups:
- largest, 64 types
- Anogenital cancers
α
HPV Five genotype groups:
- >50 types
- Nonmelanoma squamous cell carcinomas
β
HPV Five genotype groups:
- Benign diseases
y, μ, and v
- dsDNA virus
- BK - renal diseases
- JC -Progressive Multifocal Leukoencephalopathy/ PML
o qPCR kits
Human Polyomavirus
- multiple species in a single analyses creatinine PCR for influenza A, B, & RSV
- melt - curve analysis (2009 influenza H1N1 virus)
- NASBA/ Nucleic Acid Sequence Based Amplification = (RSV) - Respiratory Syncytial virus
- BEAD ARRAY TECHNOLOGY = (RSV, influenza H, nonspecific A, H1, H3, Influenza B, parainfluenza 1, 2, 3, metapneumovirus)
RESPIRATORY VIRUS
- ssRNA virus w/ spike proteins, responsible for COVID - 19 pandemic
- novel coronavirus that emerged in late 2019 in Wuhan, China causing pandemic
- symptoms vary: fever, cough, difficulty breathing, w/ severe cases potentially leading to pneumonia, acute respiratory diseases syndrome (ARDS), organ failure and death
- DETECTION: RT- PCR (gold standard), qRT - PCR, rapid antigen test, antibody test
SARS COV - 2
FUNGI:
CONVENTIONAL?
SMEAR, MICROSCOPY, CULTURE
FUNGI:
MOLECULAR?
- SEQUENCING & PCR: mold
- GENE PROBES: Histoplasma, Blastomyces, Coccidioides & Cryptococcus neoformans
- BROAD RANGE PCR & SUBSEQUENT ANALYSIS: Candida, Aspergillus, Rhizopus, other zygomycetes, HIstoplasma
- REAL - TIME PCR: Aspergillus, Pneumocystis carinii
- DIRECT PROBE HYBRIDIZATION: blastomycetes, coccidiosis immitis, histoplasma capsulatum
- PFGE: yeasts
PARASITES DETECTION:
CONVENTIONAL?
MORPHOLOGY BY MICROSCOPY
PARASITES DETECTION:
MOLECULAR?
- PCR METHODS: Trypanosomes, Plasmodium, Toxoplasma, Entamoeba & Cryptosporidium
- MULTIPLEX PCR to simultaneously detect multiple parasites: intestinal parasite in stool sample
- REAL - TIME PCR: Babesia, Trichomonas microti, Encephalitozoon spp. , Microsporidia, Trichomonas vaginalis
ANTIMICROBIAL AGENTS
2 types:
- Inhibit growth
Static (Bacteriostatic/fung
Istatic)
ANTIMICROBIAL AGENTS
2 types:
- Kill organisms
Cidal (Bacteriocidal/fungi cidal)
- A single nucleotide variant in a drug
target or transport protein can result
in resistance.
o Resistant
Staphylococcus
Pseudomonas
Klebsiella spp.
RESISTANCE TO ANTIMICROBIAL AGENTS
DETECTION OF RESISTANCE:
- Gold standard for validation of phenotype
- PCR, quantitative PCR, Multiplex PCR
Molecular methods
MOLECULAR EPIDEMIOLOGY:
- Affects many unrelated individuals at the same time
- rapidly spreading outbreak
Epidemic
- disease that sweeps across a WIDE geographical area
Pandemic
- acquired in a healthcare setting (hospital/clinic acquired)
Nosocomial infections
- Acquired from the actions of physicians (medical intervention/ treatment, surgical procedures/ use of medical devices
iatrogenic infections
MOLECULAR STRAIN TYPING METHODS
PLASMID ANALYSIS
PULSED-FIELD GEL ELECTROPHORESIS
RESTRICTION FRAGMENT LENGTH
POLYMORPHISM ANALYSIS
ARBITRARILY PRIMED PCR
AMPLIFIED FRAGMENT LENGTH POLYMORPHISM (AFLP) ASSAY
INTERSPERSED REPETITIVE ELEMENTS
INTERNAL TRANSCRIBED SPACER
ELEMENTS
SPA TYPING
MULTILOCUS SEQUENCE TYPING
MASS SPECTROMETRY
