1
Q
What is symbiotic relationship?
A
- Hosts and microbes live together long term
- indigenous microbiota
2
Q
What is a commensalistic relationship?
A
- No benefits or harm to either organism
3
Q
What is a mutualistic relationship?
A
Both host and microbes benefit
4
Q
What is a parasitic relationships?
A
Microbes cause harm to host
5
Q
What is a microbiome?
A
The collection of microorganisms that exists on the body
6
Q
What are the three types of symbiotic relationship?
A
Commensalistic
Mutualistic
Parasitic
7
Q
What does the relationship within our indigenous microbiota exist through?
A
- Co-evolution
- co-adaption
- co-dependency
8
Q
What needs to happen for a micrograms to survive?
A
- Needs to stimulate a immune response
- Needs to colonize the host and acquire nutrients
9
Q
What is infectivity?
A
- Organisms ability to establish an infection
- “contagious”
10
Q
What is pathogenicity?
A
- Ability of an organism to cause disease
- qualitative trait
11
Q
What is virulence?
A
-Extent of damage and pathology caused by an organism when it infects a host
-quantitative trait
12
Q
How does virulence factors increase an organisms pathogenicity?
A
① Organisms ability to establish itself on or in the host
② invade or damage host tissue
③ evade host-immune response
13
Q
Describe endotoxins
A
- The lipid A portion of LPS in gram-negative cell walls
- powerful stimulator of cytokine release
- complement activator, which forms anaphylatoxins C3a and C5a, which causes vasodilation and increased vasopermeability
14
Q
Describe exotoxins
A
- Potent toxic proteins released from living bacteria (mostly gram-positive)
- neurotoxins, cytotoxins, enterotoxins
- known as most potent molecules to harm living organisms
- extremely immunogenic
- induce production of protective antibodies
- may act like superantigen
15
Q
What is virulence factor?
A
Bacterial properties that determine whether an organism is pathogenic and able to cause disease
16
Q
What is necessary for organisms to be pathogenic?
A
-needs to possess genetic determinants that allow for production of either the structural components or the extracedular products that contribute to it virulence
17
Q
What are plasmids?
A
- self-replicating extra-chromosomal DNA molecules that are located in the bacteria’s cytoplasm
- contain limited number of genes
18
Q
What are bacterial cells classified as?
A
Prokaryotic cells
19
Q
What are human cells classified as?
A
Eukaryotic cells
20
Q
What are the differences between prokaryotic and eukaryotic cells?
A
- P → bacterial chromosomes are found in bacterial cytoplasm. E→ enclosed in membrane-bound nucleus
- P→ bacteria cell wall is outermost feature. E → plasma membrane is outermost feature -
- P → cell wall contains alototenymes
21
Q
What is the primary component that provides shape and rigidity of bacterial cells?
A
Peptidoglycan
22
Q
What are some important components of prokaryotic cells that affect virulence factor?
A
- Pili → adherence to host cells, resistance to phagocytosis
- flagella → adherence to host cells; motility
- capsule→ block phagocytosis, antibody attachment, C’
23
Q
What are the two variations of the bacterial cell wall?
A
- Positive gram
- negative gram
24
Q
Which variation of bacteria contains lipopolysaccharide layer (LPS)?
A
Gram-negative
25
What are the 3 components of LPS?
- outer core polysaccharide
- inner core polysaccharide
- lipid A
26
How does the capsule of bacteria contribute to the organisms ability to resist innate and adaptive immune responses?
① block the attachment of antibodies
② inhibit activation of complement
③ act as a decoy when capsular material is released into the surrounding host environment
27
What is the most important feature of the capsule?
Blocking phagocytosis by WBCs
28
What cytokines are produced by endotoxins?
IL-1
IL-6
IL-8
TNF
PAF (produces prostaglandins and leukotrienes)
29
What are two defenses against bacteria ?
Innate defenses
Adaptive defenses
30
Describe innate defenses against bacteria
- intact skin and mucosal surfaces serve as structural barriers
- antibacterial defense peptides are lysozyme, defensins, ribonuclease
- complement proteins, cytokines, acute-phase reactants
- recognition of PAMPs by PRRs such as TLRs
31
What is the function of a lysozyme?
Destroys the peptidoglycan found in the cell wall of bacteria
32
What is the function of a ribonuclease?
Destroys RNA and have antimicrobial and antiviral activities
33
What are the 3 classes of defensins?
Alpha
Beta
Theta
34
Describe alpha defensins
- produced by neutrophils, certain macrophages, and paneth cells in the small intestine
- disruptthe microbial membrane
35
Describe beta defensins
- Produced by neutrophils, and epithelial cells lining the bronchial tree and genitourinary system
- increased resistance of epithelial cells to colonization
36
What is PAMPs?
- Structural patterns consisting of carbohydrates, nucleic acids or bacterial peptides
- recognized by pattern recognition receptors (PRRs) expressed on cells of the innate immune system
37
What occurs when PRR engages with appropriate PAMP?
① triggers release of immune mediators such as cytokines and chemokines
② boosts production of various defensins and proteins
③initiates phagocytosis
38
Describe adaptive defenses for bacteria
- antibody production→ binding C’, osponization, neutralization of bacterial toxins
- cell-mediated immunity → CD4 Tcells produce cytokines that induce inflammation; cytotoxic T lymphocytes attack host cells that contain intracellular bacteria
- uses granulosomes to prevent spread
39
What is the main defense against bacteria in adaptive immunity?
Antibody production
40
What is the binding of antibodies to invading bacteria referred to as?
Osponization
41
What mechanisms do bacteria use to inhibit the NE immune response?
① avoiding antibody
② blocking phagocytosis
③ inactivating the complement cascade
④ blocking digestion
⑤ inhibiting chemotaxis
⑥ cleaving IgA
42
What mechanisms do microorganisms use to resist digestion?
- Block fusion of lysosome granules with phagosomes after being engulfed by the phagocyte
- production of extra cellular products after bacteria is phagocytized
- block action of complement
43
What are some laboratory detection tests of bacterial infection?
① culture of causative agent
② microscopic examination
③ detection of bacterial antigens
④ molecular detection of bacterial DNA or RNA
⑤ serology
44
Describe cultures of causative agent
- Grow on broth or solid media
- major means of diagnosis, but may take time or may not be possible
45
Describe microscopic examination for bacteria
- Uses gram stain or special stains
46
What tests are for detection of bacterial antigens
rapid testing done by:
- ELISA
-LFA
-LA
47
Describe molecular detection of bacterial DNA or RNA
- Can obtain results in a few hours with PCR
48
Describe serology testing for bacteria
- Detects antibodies to bacterial antigens
- to detect and confirm when options are not available
- to diagnose infections with nonspecific symptoms
- current infection indicated by IgM,high titer of IgG, or fourfold risein antibody
- to determine a past exposure to an organism
- to assess reactivation or re-exposure
49
What do the results of a serology test look Iike when a past exposure to an organism is present?
IgM-
IgG+
50
What are the disadvantages of serology testing for bacterial infection?
- Delay between start of infection and production of antibodies
- low antibody production by immunosuppressed patients
51
What are disadvantages of culturing for causitive agents?
- Several bacterial pathogens that don't have cultures available
- may take too long
- some organisms are difficult to grow
- some organisms present a danger to start it handled improperly
52
Describe gram stain
- Gram-positive bacteria stains purple with crystal violet
- gram negative bacteria stains pink with safranin
53
Why are LA and LFA assays advantageous in bacterial antigen defection?
- easily performed
- low cost
- rapid turnaround time
54
What is the most widely known molecular technology?
PCR
55
Describe streptococci structure
- are gram positive bacteria
- spherical, ovoid or lancet shaped organisms that are catalase negative and are often seen in pairs or chains
- divided into groups or serotypes on the basis o certain cell wall components, including two major proteins known as M and T proteins
- interior to the protein layer is the group-specific carbohydratethat divides streptococci into 20 defined groups, designated A-H and K-V ( known as Lancefield groups
- some strains possess a hyaluronic and capsule outside of cell wall
56
Describe streptococci
- Transmitted person to person
- additional virulence factors include exoantigens or exotoxins, proteins are secreted by the bacterial cells as they metabolizes during the course of streptococcal infections
- detection of host antibodiesto exotoxins is important in the diagnosis of sequalae such as glomerulonephritis and acute rheumatic fever
57
In streptococci, What exoantigens or exotoxins are antibodies produced for?
- Streptolysin O
-DNase B
- hylauroniadase
- nictinamide adenine dinucleotidase
- streptokinase
58
What is another name for s. pyogenes?
Group A streptococcus (GAS
59
Describe serotyping for GAS
- Used to identify a particular strain of GAS
-involves ID of the M protein antigens by precipitation with type-specific sera
60
What are disadvantages of serotyping?
- Limited availability of typing sera
- new M proteins do not react with anti-sera
- difficulty interpreting results
61
What is the most common and ubiquitous pathogenic bacteria?
GAS
62
What are virulence factors of GAS?
L-The M protein
- has net-negative charge at the amino terminal end that helps to inhibit phagocytosis
- M protein limits deposition of C3 on the bac terial surface (diminishing complement activation)
63
What are clinical manifestations of pus-forming acute group A streptococcal infection (GAS)?
- Pharyngitis (“strep throat”)
- pyoderma (impetigo or skin infection)
64
What are the clinical manifestations non-pus forming group A streptococcial (GAS) infection?
- scarlet fever
- toxic shock syndrome
- necrotizing fasciitis,
65
What is non-pus forming strep A treated with?
Antibiotics
66
What are the two major sites of strep A infection?
- Upper respiratory tract
- the skin
67
What are the symptoms of pharyngitis?
- Fever
- chills
- severe sore throat
- headache
- tonsillar exudates
- petechial rash on the soft palate, and
- anterior cervical lymphadenopathy
68
Describe impetigo
- Most common skin infection
- vesicular lesions on extremeties that become pustular and crusted.
69
Besides lesions, what are other symptoms of impetigo?
- Otitis media
- erysipelas
- cellulitis
- puerperal sepsis
- sinusitis
70
Describe Scarlet fever
- Usually associated with pharyngeal infections
- initially appears on the neck and chest then spreads over the body
- results from infection with strep Athat elaborates SpeA and SpeC ( can act as superantigens
71
What are symptoms of scarlet fever?
- Fever of 101°F or higher
- nausea
- vomiting
- headache
- abdominal pain
- rash
72
Describe necrotizing fasciitis
- May occur when Strep A
- such infection invades the muscles in the extremities or the trunk
- exotoxins produced by s. pyogenes cause rapid infection deep in fascia
73
Describe sequelae
- Conditions that are consequence of a previous disease or injury.
- results from the host response to infection
74
What are the two serious sequelae that could be developed from strep A?
- Acute rheumatic fever
- poststreptococcal glomerulonephritis
75
Describe acute rheumatic fever
- develops 1-3 weeks after pharyngitis or tonsillitis in 2% to 3% of infected individuals
-most likely caused by immune responses to streptococcal antigens that cross-react with human heart tissue
- only follows upper respiratory tract infections
76
Describe post streptococcal glomerulonephritis
- May follow strep infection of the skin or pharynx
- damages glomeruli
- deposits of immune complexes containing streptotococcal antigens in glomeruli
- characterized by damage to the glomeruli in the kidneys
- impaired because glomerular filtration rate is reduced
77
What are the symptoms acute rheumatic fever?
- Fever
- joint pain
- inflammation of heart
78
What are the symptoms of post streptococcal glomerulonephritis?
- Hematuria
- proteinuria
- edema
- hypertension
- malaise
- backache
- abdominal discomfort
- impairmentin renal function
79
What laboratory tests are run for suppurative strep A?
- Culture on sheep blood agar
-rapid assays to detect strep A
80
Describe culture on sheep blood agar for suppurative strep A.
- Small translucent colonies surrounded by clear zone of beta hemolysis
81
What are the rapid assays used to detect suppurative strep A?
- Strep screen
- LFA ( largely replaced EIAs and LA asssay)
- assays are easy to perform
- allow for single sample testing
- more sensitive
- require 2-5 minutes of hands on time
- cultured should be ran if rapid assays are negative
82
Describe serological detection of strep A sequelae
Antistreptolysin O (ASO) test
- classic hemolytic method for determining the ASO tier was the first test developed to measure streptococcal antibodies
- based on the ability of patient antibodies to neutralize the hemolytic activity of streptolysin O
-an ASO titer greater than 166 Todd units is considered a positive test
83
What are the serological detection test of nonsupprative strep A
- Antistreptolysin O (ASO)
- anti-DNase B
- streptozyme test
84
Describe antistreptolysin O (ASO) test of nonsupprative strep A
- nephelomatic methods currently used that measure ' light scatter produced by immune complexes containing streptolysin antigen
- titer is elevated in 85% of patients with acute rheumatic fever
- does not increase in patients with skin infections
- automatic and rapid
-titer increases within 1-2 weeks after infection and peaks between 3 to 6 weeks after initial symptoms
85
Describe anti-DNase B test of nonsuppurative strep A
- Produced by both rheumatic fever and impetigo patients
- tested by EIA and nephelomatic methods
- useful in patients suspected of having glomerulonephiritis preceded by skin infection
- highly specific for strep A
- if anti-DNase antibodies are present→ they neutralize reagent DNase B, which prevents it from depolymerizing DNA
- tubes are graded by color, 4+indicating intensity of color unchanged and 0 being complete color loss
86
Describe the streptozyme test
- A slide agglutination screening test for the detection of antibodies steptococoal antigens
- sheep RBCs are coated with the 5 antibodies
- hemagglutination is a positive test result
- positive in 95% of patients with acute phase post streptococcal glomerulonephritis
- Detects antibodies to five streptococcal products:
-ASO
-anti-hyaluronidase (AHase)
-anti-streptokinase (ASKase)
-anti-nicotinamide-adenine dinucleotide ( anti-NAD)
-anti-DNase B
87
What are some disadvantages of the streptozyme test?
- the least reproducible test for antibodies
- more false negatives and false positives have been reported
88
Describe heliobacter pylori
- gram-negative microaeorphilic spiral bacterium with flagella'
- transmission likely by fecal-oral route
- major cause of gastric and duodenal ulcers
- can survive in acidic environment of stomach because of production of urease, which provides a buffering zone around the bacteria
- treatment with antibiotics and anti- ulcers
-is entreated, can lead to gastric carcinoma or mucosa- associated lymphoid tumors
- resides in mucus layer, mythe gastric epithelium cells
89
What are detection tests of heliobacter pylori?
- Detect urease in stomach biopsy (CLO test)
- urea breath test
- heliobacter pylori antigens
- heliobacter pylori antibodies
-ELISA is method of choice!
-IgG in serum (indicates active infection
-titer decreases after successful treatment
90
What are virulence factor of H. Pylori?
- Production of the protein CagA (highly immunogenic)
- vascuolating cytotoxins or VacA
91
What is the most specific test to detect H. Pylori?
Culture
92
Describe the urea breath test
- Patient ingests urea labeled with radioactive carbon (14^C) or nonradioactive (13^C) is broken down by the urease enzyme of H. pylori, producing ammonia and bicarbonate
- excellent sensitivity and specificity
93
What test is usually done because of potential of treatment failure for H.pylori?
- Analysis of stool samples before and after antimicrobial therapy for H. Pylori antigens is done to determine if the bacteria have been eliminated following treatment
94
An H. Pylori infection results in production of what?
IgG (test detect this class)
IgA
IgM
95
What is considered a successful treatment of H. Pylori infection?
Decrease in antibody titer more than 25%
96
Describe mycoplasma pneumonia
- tiny bacteria that lack a cell wall
- leading cause of respiratory infections
- spread by respiratory droplets
- belong to class Mollicutes
- incubation period is 1 to 3 weeks
- infection has insidious onset
97
What are the symptoms of a mycoplasma pneumonia?
- fever
- headache
- malaise and
- cough
98
What are clinical manifestations of mycoplasma pneumoniae?
- Walking pneumonia
- raynaud syndrome
- stevens-Johnson syndrome
99
What is a mycoplasma?
- Represents the smallest known free-living life forms
- have small genome
100
Describe stevens-johnson syndrome
- Condition which top of skin dies and sheds -
101
Describe raynaud syndrome
- transient vast spasm of the digits in which the fingers turn white when exposed to cold
- may be related to the development and action of cold agglutinins in body
102
What are laboratory diagnosis procedures for M. Pneumonia?
- Culture
- antibody detection
- cold agglutinins
- molecular methods
103
Describe a culture of M. Pneumoniae
- Gold standard but isn't used anymore
- produces mulberry colonies with a “fried egg" appearance on specialized media
104
Describe antibody detection for M. Pneumoniae
- Most useful diagnostic assay
- IgM → recent-infection
- IgG → possible reinfection
- most widely used → EIAs
105
Describe cold agglutinin testing of M. Pneumonia
- Present in 50% of patients with m. Pneumonia but not specific for the infection
- cause RBC agglutination at 4°C
- reversible at 37°C
- not very specific or sensitive
106
Describe molecular methods of m. Pneumonia
- Will most-likely become gold standard
- LAMP technology
107
Describe rickettsial infections
- Obligate intracellular gram-negative bacteria
- spotted fever groups (spf) → Rocky Mountain spotted fever
- typhus group (tg) → epidemic typhus
- organisms transmitted by arthropods (ticks,mites, lice or fleas) through biting after feeding on an infected animal
108
Describe Rocky Mountain spotted fever (RMSF)
- Caused by R. Rickettsii
- transmitted by 3 species of ticks
109
What are the symptoms of RMSF?
- Headache
- nausea
- vomiting
- diarrhea
- skin rash (usually starts on hands and soles of feet and proceed to trunk)
- death
* occurs 2-14 days after tick bite
110
How is RMSF diagnosed?
- Clinical presentation of symptoms
- serology by IFA (gold standard)
111
How do RMSF organisms spread in the body?
- Via lymphatic and circulatory system
- attach to and invade target cells (vascular endothelium)
- organism multiply by binary fission
112
What is the main pathophysiological event caused by RMSF?
Endothelial cell damage which leads to increased vascular permeability, resulting in edema, hypovolemia, hypotension, and hypoalbuminemia
113
What is RMSF treated with?
Doxycycline
114
An ASO titer and a streptozyme test are performed on a patients serum. The ASO titer is negative, the streptozyme test is positive, and both the positive and negative controls react appropriately. What can you conclude from these test results?
The patient has an antibody to a streptococcal exoenzyme other than streptolysin O
115
Why is serological identification of a current infection with helicobacter pylori difficult?
Antibodies remain after initial treatment
116
What is the principle of the IFA test for detection of antibodies produced in RMSF?
① specific antibodies in serum sample attach to the antigens fixed to a microscope side
②attached antibodies are stained with fluoroscein- labeled anti-human immunoglobulin and visualized with a fluorescence microscope
117
What is the principle of LFA?
- involves movement of a liquid sample containing ' the analyte of interest along a strip that passes through various zones containing labeled antibodies specific to analyte
- if antigen-antibody complex is present, it is captured by another antibody at the end of the strip, resulting in a visible line
118
What are spirochetes?
- Long, slender, helically coiled bacteria containing axial filaments or periplasmic flagella
- wind around the bacterial cell wall and are enclosed by an outer sheath
- gram- negative
- microaerophilic
- corkscrew flexion and motility
119
Describe spirochete diseases
- Caused by spirochete bacteria
- localized skin infection, disseminates to numerous organs as disease progresses
- cardiac and neurological involvement if disease remains untreated
120
What are the two major spirochete diseases?
Lyme disease
Syphilis
121
Describe syphillis
- Sexually transmitted disease caused by treponema ' pallidum
- rapidly destroyed by heat, cold, and drying
- direct contact with open lesion needed
- transmission to fetus during pregnancy
- bloodborne transmission rare
- most common spirochete disease
- have TROMP membrane proteins
122
What are the pathogens in the pallidum subspecies?
- T. Pallidum subspecies pertenue
- T. Pallidum subspecies endemicum
- T. Carateum
123
Describe T. Pallidum subspecies pertenue
- Agent of yaws
- found in tropics
124
Describe T. Pallidum subspecies endemicum
- Cause of nonvenereal endemic syphillis
- found in desert regions
125
Describe T. Carateum
- The agent of pinta
- found in central and South America
126
What are the stages of syphilis?
① primary stage
② secondary stage
③ latent stage
④ tertiary stage
127
What is a characteristic of the primary stage of syphilis?
- Development of chancre
- this stage lasts 1-6 weeks
128
What are symptoms of the secondary stage of syphillis?
- Generalized lymphadenopathy
- malaise
- fever
- pharyngitis
- rash
129
Describe the latent stage of syphillis?
- Asymptomatic
- follows disappearance of secondary stage
- patients are not infectious at this time with the exception of pregnant women
130
What are symptoms of the tertiary stage of syphilis?
- gummatous
- cardiovascular
- neurosyphilis
- Tabes dorsalis (shuffling gate)
131
What are occurs in the primary stage of syphilis when contact has been made with susceptible skin site?
- Endothelial cell thickening occurs with aggregation of lymphocytes, plasma cells and macrophages
- chancre occurs 10-90 days after infection
132
What stages of syphilis could spontaneous healing occur?
Primary
Secondly
133
Describe tertiary stage of syphilis
- stage occurs most often 10-30 years following the secondary stage
134
What are gummas?
- Localized areas of granulomatous inflammation that are most often found on bones, skin or subcutaneous tissue
- contains lymphocytes, epithelioid cells, and fibroblasts
- may heal spontaneously with scarring
135
Describe cardiovascular complications in the tertiary stage of syphilis
- involve ascending aorta
- symptoms caused by destruction of elastic tissue in the aortic arch
136
What is the treatment for syphilis?
Antibiotics such as penicillin
137
Describe congenital syphilis
- Occurs when a woman who has early syphilis or early latent syphilis transmits treponemae’s to the fetus
- most affected in second and third trimester
- causes death in 10% of cases
138
What are the symptoms in a newborn that has congenital syphilis?
- Live born infants may asymptomatic at birth but develop symptoms later
- runny nose
- skin rash
- generalized lymphadenopathy
- hepatosplenomegaly
- jaundice
- anemia
- bone abnormalities such as saddle nose or saber shins
- neurosyphilis
139
What are laboratory diagnostic procedures of syphilis?
Direct detection
- demonstration of treponema's in active lesions
- dark-field microscopy
- fluorescent antibody staining
Serological test
- nontreponemal
- treponema
140
How can primary and secondary syphilis be diagnosed?
Presence of T. Pallidum in exudates from skin lesions
141
When do false negatives occur in dark-feld microscopy when testing for syphilis?
① a delay in evaluating the slides
② insufficient specimen obtained
③ patient is pretreated with antibiotics
142
Describe fluorescent antibody testing for Syphilis
- Sensitive
- highly specific
- direct and indirect methods available
- No live specimen required
143
What testing should be performed for syphilis if no lesions are present?
Serological
144
Describe nontreponemal tests
- detect antibody against cardiolipin (Reagin)
-sensitive, inexpensive, and simple to perform
- includes:
-venereal disease research laboratory test (VDRL)
-rapid plasma Reagin test (RPR)
-both→ look for flocculation
-screen →undiluted patient serum
-titer→test twofold dilutions of patient serum
145
Describe VDRL test procedure
① patient serum mixed on slide with cardiolipin- lecithin-cholesterol antigen suspension
② rotated for 4 minutes at 180 RPM
③ viewed under a light microscope for flocculation
- results:
-reactive→ medium to large clumps
-weakly reactive → small clumps
-nonreactive → no clumps or slight roughness
146
What is Reagin?
- A lipid released from membranes of cells damaged as a result of the infection
147
Describe the RPR test procedure
①patient serum mixed on a card with charcoal particles coated with cardiolipin antigen
② rotate 8 minutes, 100 rpm
③ observe for macroscopic flocculation
148
Briefly describe VDRL
- quantitative and qualitative slide flocculation
- all sera that is weakly reactive and reactive must be retested using quantitative slide test
149
Why are patients in the secondary stage of syphilis subject to a false positive?
Prozone phenomenon ( antibody excess)
150
Briefly describe RPR
- Cardiolipin- containing antigen suspension is bound to charcoal particles, making resteasier to read
- all reactive tests should be confirmed by retesting using doubling dilutions in a quantitative procedure
- more sensitive than VDRL in primary stage of syphilis
151
Describe treponemal tests for detecting syphilis
- detect antibody to T. Pallidum
- usually positive before nontreponemal tests
- lack of sensitivity in congenital syphilis and neurosyphilis
- fluorescent treponemal absorption (FTA-ABS)
- T. Pallidum particle agglutination (TP-PA)
- automated immunoassays:
-enzyme-linked immunosorbent assay (ELISA)
-chemiluminessent immunoassays (CLIA)
-multiplex flow immunoassays (MFI)
152
Briefly describe FTA-ABS
-an indirect immunofluorence test antibody to T. Pallidum
- use Reiter strain
-false positive results may occur in patients with SLE
153
Describe FTA-ABS test procedure
① patient serum is incubated with sorbent (an extract of nonpathogenic treponemes) to remove cross-reacting antibodies
② absorbed patient serum is incubated with a microscopic slide fixed with T. Pallidum (Nichol’s stain)
③ following a Wash step, anti-human Ig conjugated with fluorescein is added
④ after a second incubation and wash, the slide is examined under a fluorescent microscope
*no fluorescence indicates a negative test, and a result of 2+ or above is considered reactive
154
Describe TP-PA test procedure
- Patient serum and controls are diluted and incubated with unsensitized gel particles or gel particles sensitized with T. Pallidum
- positive → agglutination (smooth mat covering surface of well)
- negative→ no agglutination (button)
155
Briefly describe TP-PA test
- Used colored gelatin particles coated with treponemal antigens
- more sensitive in detecting primary syphilis
156
What type of EIAs are used to detect the antibodies to T. Pallidum?
- one or two-step sandwich assays
- one step competitive assays
- immune capture assays
157
Describe sandwich assays of syphilis
- Antibodies in the patient sample bind to recombinant T. Pallidum antigens coated onto microtiter plate wells
158
Describe the immune capture of syphilis
- Micrometer wells are coated with antibody to IgM or IgG and are reacted with patient serum
159
What are capture EIAs useful at diagnosing?
Congenital syphilis
160
Describe competitive EIAs of syphilis
- treponemal antibody in the patient sample competes with an enzyme- labeled treponemal antibody conjugate for T. Pallidum antigens bound to microtiter plate wells
161
Describe CLIAs of syphilis
- one step sandwich assay→
- higher sensitivity, faster performance, and more stable reagents
- patient sampleis incubated with paramagnetic microparticles that have been coated with T. Pallidum antigens linked to chemiluminescent derivative
162
Describe PCR testing of syphilis
- involves isolating and amplifying a specific sequence DNA
- extremely sensitive (highest in primary syphilis), more than dark field microscopy
- availability is limited
163
What specimens can be used to detect treponemes in PCR?
- Whole blood
- spinal fluid
- amniotic fluid
- various tissues
- swab samples from lesions
164
What is the main disadvantage of nontreponemal testing? When does it occur?
Subject to false positives
- hepatitis
- IM
- varicella
- herpes
- measles
-malaria
- TB
- SLE
- leprosy
- intravenous drug use
- autoimmune arthritis
- advanced age
- advanced malignancy
165
What should a reactive nontreponemal test be confirmed by?
A more specific treponemal test
166
When should treponemal test for syphilis be performed?
- Confirmatory test to distinguish false positive from true positive nontreponemal results
- establish diagnosis in late latent or late syphilis
167
Why are nontreponemal tests for syphilis performed?
- screening for syphilis
- monitoring the progress of the disease
- determining the outcome of treatment
168
Describe traditional testing algorithm for syphilis
① initial screening with nontreponemal (VDRL or RPR) ''
② if reactive, perform treponemal test for confirmation (TP-PA or FTA-ABS). If nonreactive, no further testing needed
③ if reactive, patient is positive for syphilis. If nonreactive, previous positive result was false.
169
Describe reverse sequence algorithm of syphillis
-testing order reversed
① samples are screened by automated treponemal immunoassay
② it reactive, results are confirmed by nontreponemal testing
170
What are the advantages of reverse algorithm over traditional?
① cost
② automation
③ saves time
④ less errors
⑤ can detect more early, late and treated syphilis cases
⑥ high sensitivity
171
In reverse algorithm testing for syphilis, What does it mean when the initial automated test result being positive and the RPR that follows gives a negative result?
Either:
- caused by false positive result
- past syphilis infection
- day primary syphilis ( haven't produced nontreponemal antibodies yet)
* use TP-PA as secondary confirmatory test
172
What should be performed when monitoring patients syphilis?
- Perform nontreponemal antibody titer
- titers decline with successful treatment
173
What should be performed for congenital syphilis?
- Perform nontreponemal tests on mother and infant at birth
- IgM-specific treponemal assays to confirm
174
What should be performed if neurosyphilis is suspected?
- Perform CSF VDRL or ELISA on cerebrospinal fluid
175
What are the tests in order of reverse algorithim sequence?
① perform EIA or CIA
② if positive, perform quantitative RPR (or other nontreponemal tests)
③ if RPR positive,indicates past or present syphilis infection. if negative, perform TP-PA
④ iF TP-PA is positive, indicates past or present inflection of syphilis
176
What test is recommended to confirm congenital syphilis?
Western blot
177
Describe lyme disease
- Caused by the spirochete bacterium Borrelia burgdorferi
- multisystem illness involving the skin, nervous system, heart and joints
- transmitted by Ixodes ticks
- main reservoir: white-footed mouse
- most common vector-borne disease in the US
178
Describe the clinical manifestations of Lyme disease
-stage 1: localized rash and hallmark erythema migraines
-stage 2: early dissemination
-stage 3: late dissemination with arthritis, heart and nervous system involvemenr
179
What is the most prevalent neurological sign of lyme disease?
Facial palsy
180
What is underneath the outer envelope of B. Burgdorferi?
- 7-11 endoflagella or periplasmic flagella
- these nun parallel to long axis of organism
- made up of 41-kDa subunits (elicit strong antibody response)
- organism divides every 12 hours
181
What are the types of Ixodes ticks?
① Ixodes scapularis → northeast and Midwest US
② Ixodes pacificus → in the west
③ Ixodes ricinus → Europe
④ Ixodes persulcatus →Asia
182
What are peak feed ding seasons for ticks?
- late spring
- early summer
- fall
183
Describe erythema migrans
- Localized rash stage
- hallmark rash of early infection of Lyme disease
- appears 2- 14 days after bite
- begins as a small red papule where bite occurred, then radio expands to form a large ring-like erythema and central area exhibits partial clearing
- must be atleast 5 cm in diameter to diagnose with Lyme disease
184
Describe the early dissemination stage
- Occurs via bloodstream in days to weeks following rush
-skin, nervous system, heart, and joints involved
-usually migratory pain occur in joints, tendons, muscles, and bones
185
Describe late dissemination stage of Lyme disease
- Can develop in untreated patients months to years after acquiring infection -
- major manifestations→ arthritis, peripheral neuropathy and encephalomyelitis ( respond well to conventional antibody treatments)
- some develop fatigue, concentration and short term memory problems and musculoskeletal pains
186
Describe antibody response of Lyme disease
- May not be detectable for 3-6 weeks
- IgM occurs first, followed by IgG
- IgG peaks at 3rd and 4th weeks of infection
187
Using the standard testing algorithm, What tests (in order) are ran to diagnose Lyme disease?
① screened with sensitive ELISA or IFA
② it positive or borderline, western blot test for IgM is performed to confirm results of patient with symptoms present less than 30 days. western blot test for lgG is patient had symptoms that exceeded 30 days
188
What are characteristics of standard testing algorithm of Lyme disease?
- Costly
- western blot can be difficult to interpret
- highly specific
- low sensitivity for early Lyme disease
189
What are the tests (in order) performed in the modified two-tiered algorithm?
① symptomatic patients are tested with sensitive ELISA that uses purified borrelia peptide antigens
② if positive or borderline, test with different ELISA method for confirmation
190
What are characteristics of the modified two-tiered algorithm?
- Easy to perform and interpret
- comparable specificity to standard method
- increased sensitivity to detecting early Lyme disease
191
Describe immunofluorence assay (IFA) of Lyme disease
- First test used to evaluate the antibody response
① doubling dilutions of patients seem are incubated with commercially prepared slides coated with antigen from whole or processed borrelia spirochetes
② following wash step, anti-human globulin with fluorescent tag is added and reacts with any specific antibody bound to spirochetes on slide
③ after second wash, side is viewed under a fluorescent microscope
* test considered positive only if titer of 1:256 or higher is obtained
192
What can produce false positives in IFA?
- Closely related organisms can cross react
- autoimmune connective tissue diseases such as RA and SLE
- fluorescent light pattern is very subjective
193
Describe EIA testing for Lyme disease
- ELISA is inexpensive and yields timely results
- automation
- high volume testing
- less sensitive in early Lyme disease
194
What can cause false positives with EIAs when testing for Lyme disease?
- syphilis or other treponemal diseases '
- yaws
- periodontal disease
- relapsing fever
- leptospirosis
- IM
- RMSF
195
Describe western blot testing for Lyme disease
- Also called immunoblotting
- very complex
- patient scrum is incubated with nitrocellulose membrane containing electrophoresed B. burgdorferi antigens
- positive IgM results →2 of 3 characteristic bands
- positive IgG results →5 of 10 characteristic bands
196
Describe PCR testing of Lyme disease
- involves Probes for Target DNA that is present only in B. Borrellia
- involves extraction of DNA, followed by amplification using specific primers, DNA polymerase and nucleotides
- highly specific
- skin biopsy is not justifiable
- used to diagnose difficult neurological and arthritic cases
197
Describe treatment of Lyme disease
- Antibiotics
- oral doxycycline is first treatment of choice in early Lyme disease who are not pregnant
198
Describe Leptospirosis
- Caused by leptospira species '
- zoonotic infection associated with occupational and recreational activities
- humans are exposed by mucous membrane contact with urine-contaminated water
- causes febrile episode that can progress to severe disease involving renal, liver, pulmonary and CNS
199
What are characteristics of the leptospira species?
- Thin, flexible, and lightly coiled spirochetes
- pointed ends with hooklike shape
200
Describe the stages of leptospirosis
- incubation period→ 2-30 days
- usually manifests 5 -14 days after exposure
- severe cases can lead to Weil’s disease
201
What are symptoms of leptospirosis?
- Febrile episode of headache
- myalgia
- nausea
- vomiting
- diarrhea
- renal involvement
- hepatic involvement
- pulmonary involvement
- CNS involvement
- renal failure
- jaundice
202
What are the laboratory tests performed for leptospirosis?
- Specimens→ blood or serum for first week, after that, urine or serum
- IgM screening by ELISA, immunoDOT, and LFA
- MAT is gold standard for confirmation
203
What is the treatment for leptospirosis?
Antibiotics
204
Describe the presence IgM and IgG in Lyme disease
- Cannot be detected until 3-6 weeks after symptoms appear
- two-tiered method is used (IFA or EIA screening, western blot or different EIA for conformation
205
What is the purpose of the Reiter treponemes in FTA-ABS?
- Prevents cross-reactivity with antibody to non-pathogenic treponemes
206
What test is recommended for testing cerebrospinal fluid for detection of neurosyphilis?
VDRL
207
What are the advantages of direct fluorescent antibody testing to T. Pallidum?
- Reading is less subject
- monoclonal antibody makes the reaction very specific
-Slides can be prepared for later reading
208
What syphilis test detects specific treponemal antibodies?
FTA-ABS
209
An RPR test done on a 19-year old woman as part of a prenatal workout was negative but exhibited a rough appearance. What should be done next?
- Make serial dilutions and do a titer
210
What test can be used to distinguish between IgG and IgM antibodies in syphilis?
Treponemal EIAs
211
A 24 -year old man who had just recovered from IM had evidence of genital lesions. His RPR was positive. What should be done next?
- Do a confirmatory troponemal test
212
What is toxic shock syndrome associated with??
Overproduction of cytolanes
213
What are ANTIGEN noninvasive tests used to detect H. Pylori?
- Urea breath test
- ELISA
- LFA
- DNA
214
What antibody is associated with M. Pneumoniae?
Anti-I (cold agglutinin)
215
What disease is associated with anti-i?
Hemolytic anemia
216
What is a rapid urease method for H. Pylori?
CLOtest
MLT immunology
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