(F) Advances of DX Parasitology: (lecture-based) Flashcards
Rupert-based
- Gold standard
- Allows detection of parasites
- Morphologically distinct parasites are differentiated
- Requires high parasite density to achieve a good level of sensitivity
MICROSCOPY
Enumerate the Basic Microscopic Techniques (5)
- DFS
- Kato Katz
- Formol-Ether-Sedimentation Technique
- Harada-Mori
- Mcmaster technique
TOF. For a good level of sensitivity, there should be a low parasite densitive.
F (high parasite density)
2 Reagents used for DFS
Lugol’s iodine and NSS
DFS
TOF. The lugol’s iodine is ONLY used to improve the nuclear detail and visualize the morphology of egg and ova.
F (not only, it is also used for larvae or other diagnostic feature/stage depending on the parasite accd. to ser)
Kato Katz Technique, except:
A. Determine the intensity of infection
B. Quantitative method
C. 6.5mm template hole size
D. Green cellophane tape is used
E. Read under the microscope using OIO
E (10x and 40x obj. lang)
Arrange in Order
Kato Katz Technique
A. Place cellophane tape
B. Sieve the stool in a mesh screen
C. Place the stool in a template
D. Wait for 10-30 minutes
E. Read under 10x and 40x objective
CBADE
Here’s the complete:
- Stool sample, then place the stool in a template ○ Template contains a hole where the stool sample is placed ○ 6.5 mm (diameter of hole)
- Spatula is needed
- A sieve/screen is used where the stool is sieved in a mesh screen
- Cellophane tape is placed
- Wait 10-30 minutes.
- Read under the microscope using 10x and 40x power objective
This technique is used to homogenize the stool sample and form 4 layers after centrifugation.
Formol Ether Sedimentation
- Homogenize the stool sample and take a quantity of 1g
- Use 10 ml of 10% formol water solution to emulsify the stool and filter the mixture with a sieve and gauze to remove large particles
- Transfer 7 ml of the filtered solution into 15 mL centrifuge tube and add 3 ml of ether or ethyl acetate
- Centrifuge the mix at 1600 rpm at 10℃ for 5 minutes
- After centrifugation, there should be 4 layers
- Discard the supernatant and add 1 ml of sediment onto a glass slide, and read it under the microscope with 10x and 40x objectives
FEST
- Emulsify the stool
- Remove large particles
- Promote separation fo the sediment from the supernatant
A. NSS
B. 10% formol water
C. Sieve
D. Centrifugation
BCD
FEST
Enumerate the 4 layers in order
upper to last layer
Ether, Fecal debri, Formol Water, Sediment (containing the parasites)
FEST
Arrange in Order
A. Centrifuge the mix at 1600 rpm at 10℃ for 5 minutes
B. Formation of 4 layers
C. Discard the supernatant and add 1 ml of sediment onto a glass slide
D. Take 1g of homogonize stool sample
E. Use 10 ml of 10% formol water solution to emulsify the stool and filter the mixture with a sieve and gauze to remove large particles
F. Transfer 7 ml of the filtered solution into 15 mL centrifuge tube and add 3 ml of ether or ethyl acetate
DEFABC
- Filter paper strip with stool sample
- Remove the strip and close the tube
- Centrifuge the sample at 1500 rpm for 2 minutes
- Discard the supernatant
- Using a pipette, remove the sediment, place it on a slide and re-cover it with a cover glass
- Read the slide with the 10x power objective of the microscope
Harada Mori
A. Allows visualization of hookworm
B. Allows visualization of Strongyloides stercoralis filariform larvae
C. Allows visualization of Angiostrongylus stercoralis
D. AB
E. BC
D
harada mori
identify
* Incubation days
* Centrifuge (RPM for how many minutes)
- 3-7; pero p’wede rin 10 days
- 1500rpm for 2 minutes
Harada Mori
- For examining animal parasites
- Uses ZnSO4 solution but also uses Sucrose solution in some
- Read with 10x microscope objective
- Mimics hemocytometer
- Checks for the presence of eggs
MCMASTER TECHNIQUE
McMaster Technique
A. Add 60 ml of ZnSO4 solution in a beaker and homogenize
B. Filter the mixture into another beaker
C. Transfer filtrate into a clean 15 ml tube and place a cover glass on the top of tube for 15 min
D. Fill both McMaster chambers, mount the chambers on a microscope and examine with the 10x microscope objective
E. Carefully transfer the cover glass onto a glass slide and read with 10x microscope objective
ABCED
- Weigh 2g of stool
- Add 60 ml of ZnSO4 solution in a beaker and homogenize (vigorously by vortexing)
- Filter the mixture into another beaker
- Transfer filtrate into a clean 15 ml tube and place a cover glass on the top of tube for 15 min
- Carefully transfer the cover glass onto a glass slide and read with 10x microscope objective
- Fill both McMaster chambers, mount the chambers on a microscope and examine with the 10x microscope objective
Power Objective
- Kato Katz
- FEST
- Harada-mori
- Mcmaster technique
A. 10x
B. 40x
C. both
D. neither
may repeating answers
CCAA
INTESTINAL NEMATODE INFECTIONS
Microscopic techniques used for diagnosis
- DFS and kato-katz
INTESTINAL NEMATODE INFECTIONS
- Used to increase diagnostic sensitivity of microscopy; labor-intensive have poor
reproducibility owing to operator error, and usually require centrifugation steps - Enables assessing the prevalence and intensity of infection in nematode control programmes
identify techniques
- Floatation and conc techniques
- Kato katz
INTESTINAL NEMATODE INFECTIONS
TOF. Larvae of Ascaris lumbricoides may be observed in the sputum suring pulmonary migration.
T
INTESTINAL NEMATODE INFECTIONS
Advantage
A. Sensitive
B. ID of nematode eggs
C. ID of larvae and adult forms of nematodes
D. AB
E. BC
C (the others are the disadvantage; make it opposite)
INTESTINAL NEMATODE INFECTIONS
TOF. Microscopic analysis of multiple samples with multiple slides per sample over several days is required for sensitive detection and quantification of nematode infections.
T
INTESTINAL NEMATODE INFECTIONS
Familiariza yourself with the advantages and disadvantages
OK
pati na rin intestinal tapeworm infections + liver lung and blood flukes infection advantages and disad
INTESTINAL NEMATODE INFECTIONS
TOF. The DFS as the most commonly used microscopic technique, enables assessing the prevalence and intensity of
infection in nematode control programmes
F (kato katz)
INTESTINAL NEMATODE INFECTIONS
TOF. In the Kato-Katz method, nematode eggs are observed at specific intervals (“clearing times”) after slide preparation, affecting the test’s sensitivity and specificity for hookworm detection. Hookworm eggs, with their thin shells, may disintegrate over time and are reported as helminth eggs.
T
INTESTINAL TAPEWORM INFECTIONS
TOF. Conventional coproscopic examination has HIGH sensitivity due to variable egg excretion in stool and the small sample volume analyzed.
F [low sensitivity; (60-70% of cases missed)]
LIVER, LUNG AND BLOOD FLUKES INFECTION
TOF. Microscopy cannot diagnose early-stage (prepatent) infections since it relies on detecting parasite eggs, which, in schistosomiasis, appear at least two months after the last freshwater contact.
T
Microscopic techniques used for diagnosis
- Kato-Katz Method
- Wet mount preparation of fresh or concentrated stool
A. INTESTINAL NEMATODE INFECTIONS
B. INTESTINAL TAPEWORM INFECTIONS
C. LIVER,LUNG AND BLOOD FLUKES INFECTION
Just choose one infection
A
Microscopic techniques used for diagnosis
- Wet mount preparations of fresh or concentrated stool
- Preianal scraping (Graham’s test)
- Haematoxylin and eosin staining of gravid proglottids obtained from stool samples
A. INTESTINAL NEMATODE INFECTIONS
B. INTESTINAL TAPEWORM INFECTIONS
C. LIVER,LUNG AND BLOOD FLUKES INFECTION
1 answer
B
Microscopic techniques used for diagnosis
- Wet mount preparation of stool/ bile/ sputum/ urine after the concentration by sedimentation or flotation techniques
- Membrane filtration technique using nucleopore filters for schistosomal infections
- Kato-Katz method ○ Sedimentation or flotation techniques
A. INTESTINAL NEMATODE INFECTIONS
B. INTESTINAL TAPEWORM INFECTIONS
C. LIVER,LUNG AND BLOOD FLUKES INFECTION
C
- Detect either antigen or antibodies in clinical specimens
- Useful in monitoring response to chemotherapy
- Alternative test; support test that we can employ to fully diagnose true positive patients with parasitive infection.
Immunodiagnosis
Familiarize ka nalang beh mga naka-include sa immunodiagnosis
15 items lang ‘to lahat sa exam
o Immunofluorescent assay (IFA)
o Enzyme linked immunosorbent assay (ELISA)
o Hemagglutination test (HA)
o Immunoblotting (dot blot)
TOF. Microscopic techniques can be unnecessary for patients hence we have immunodiagnosis for non-invasive procedures and accurate/rapid results.
T
The detection of (Antibody/Antigen)
- This test is used against the parasite in question are used when biologic specimens do not permit microscopic diagnosis during chronic or asymptomatic infections
- Indicates recent or past infections
- Are useful when significant levels of athis are produced with parasitic infections
Antibodies
TOF. In antibody detection assay, use WHOLE parasites from animal models or in vitro cultures soluble crude extracts as antigens
T
Pls study the ANTIBODY DETECTION TEST OFFERED AT CDC
okay thx
familiar urself lang raw
The detection of (Antigen/Antibody)
- Detection of specific parasite ________ is more sensitive and specific immunodiagnostic test to determine the disease status of patients
- Cross reactivity
- Feces, urine, duodenal fluid or biopsy specimens for the small intestine or urine (for intestinal parasites)
- Serum or whole blood (for blood parasites)
- To determine the immune status of the patient
DETECTION OF ANTIGEN
Detection of Antigen
Arrange in order
A. Capture antibody binds to antigen with high specificity
B. Wells are pre-coated with capture antibody and sample is added
C. Enzyme labeled antibody binds to Fc region of detection antibody
D. Primary antibody binds the immobilized antigen
E. Substrate is catalyzed by the enzyme and gives color
BADCE
Detection of Antigen
Shape of the antibody
Y
Components of Detection of Antigen
What is done for the first antibody?
either monoclonal or polyclonal
immobilized in a solid phase
Components of Detection of Antigen
What is used when it comes to this solid state
Microtiter plate and nitrocellulose membrane
Components of Detection of Antigen
The parasite antigen is detected by the second antibody, which is usually (monoclonal/polyclonal) antibody labeled with an enzyme
Mono
Components of Detection of Antigen
TOF. Always perform a positive or negative cut upp control
T
Components of Detection of Antigen
Sensitivities and specificities of commercially available kits
range from?
93 to 100%
Parasites that utilizes antigen detection assay
A. Cryptosporidium
B. Giardia
C. E. histolytica
D. A & B
E. A, B, C
E
- Provide quickest turnaround time and the least requirement for an experienced laboratory personnel
- offer the convenience of multiple results in one reaction device without the need for special equipment
- Both EIA and rapid test kits show good correlation with DFA
Rapid immunochromatographic assays
- most sensitive and specific test in the diagnosis of cryptosporidiosis and G. duodenalis
DFA
DFA uses a ________ which detects antigens on the surface of Cryptosporidium oocysts
fluorescein isothiocyanate (FITC)-labeled monoclonal antibody
- detecting the presence of an antigen
- detecting the presence of antibodies
A. DFA
B. IFA
identify each
AB
- The primary principle of this technique is when the antigen is present in a biological sample such as stool and the monoclonal antibody present in the reagent that contains your FITC it will bind to the antigen on the surface of the egg/ova of the parasite to form an antigen-antibody reaction
IMMUNOFLOURESCENCE ASSAY
- enzyme used to differentiate E. histo from E. dispar since they can’t be differentiated under a microscope
GALACTOSE ADHESIN
- Enzyme detected in E . histolytica using monoclonal antibodies in commercially available diagnostic test kits
- Very specific for E . histolytica was found to be highly sensitive and specific by several studies
- Amebic liver abscess (ALA) can be diagnosed by SEROLOGY using this enzyme
A. Techlab E . histolytica II
B. Galactose adhesin
C. Gal/GalNac Lectin
answer each bullet and onward cards
BA
COMMERCIALLY AVAILABLE PARASITE ANTIGEN DETECTION TEST
pa-study thank u po
- Detect the presence of the parasite up to the molecular level; Genetic contents for identification of specific
organisms. - This offer greater sensitivity and specificity than the previous mentioned tests compared with serological and microscopy
- Allow for direct detection of parasites in samples including those with very low parasite load from asymptomatic patients
- Uses two oligonucleotide primers which flank the parasite target sequence and Taq polymerase
MOLECULAR DIAGNOSIS
Steps in PCR
- separate double stranded DNA at high temp 94-95°C
- 50-60°C ,facilitates binding of primer to its complementary sequence
- 72-74°C DNA synthesis process
identify each step
kaya mo na yan
Denaturation, Annealing, Extension
Steps in PCR
The amplified target is then analyzed by?
gel electrophoresis
or alternatively, by ELISA methods
PCR Variants
- requires two amplification steps. 1st 15-30 cycle and 2nd round amplification, use a second set of primer to bind the internal template to first amplification to improve sensitivity and specificity.
- detect multiple species of parasite in a single sample in a single tube
- Quantify original template concentration
A. Real-time PCR
B. Nested PCR
C. Multiplex PCR
BCA
Real-time PCR
Various fluorescence, except:
A. TaqMan probes
B. SYBR Pink
C. ALL
D. NOTA
B (SYBR green)
- a DNA binding dye that fluoresces strongly when bound to double stranded DNA
- a DNA binding dye and it intercalates with the DNA that cause fluorescence strongly with bound to double stranded DNA
SYBR GRE EN DETECTION IN REAL -TIME PCR
SYBR GRE EN DETECTION IN REAL -TIME PCR
TOF. Low amplification = High fluoresce
F (low fluoresce)
Components of a SYBR green
A. dNTPs
B. Forward and reverse primer
C. SYBR green dye
D. All
D
- is designed to be complementary to a specific sequence spanned by the PCR primers
- has a reporter dye at its 5’ end and a quencher dye at its 3’ end.
- Same composition of master mix, templates, double stranded DNA, DNTPs, forward and reverse primer and probe that contains report and quencher dye and light source of instrument and resulting fluorescence that normally emitted by reporter dye
TaqMan RT-PCR
TaqMan RT-PCR
TOF. As long as the reporter and quencher dye is intact, there will be a fluorescence.
F (as long as there’s a close proximity with the probe and quencher dye, fluorescence will not happen due to the quenching effect)
TaqMan RT-PCR
This effect is the process that normally decreases the intensity of fluorescence.
Quencher dye
OTHER NEW MOLECULAR AP PROACHE S IN THE DIAGNOSIS OF PARA SITIC DIEASE
- Current trend
- Dectect multiple organism
- Achieve result in a matter of 30 mins to an hour as compare to conventional wait to several hours before process complete.
- Provide increase release of result
Loop-mediated isothermal amplification (LAMP)
+ luminex-based technologies
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
TOF. Do not require extraction of parasite DNA (as compare to conventional PCR).
T
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
TOF. Specimens of interest is mixed with diagnostic primers, substrates, and DNA polymerase capable of strand displacement in a microcentrifuge tube in MULTIPLE TUBES.
F (single tube)
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
TOF. The resulting turbidity is proportional to the amount of DNA synthesized.
T
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
Measurement
A. Naked eye
B. Real-time
C. Both
D. NOTA
C
LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)
Temperature
60-65C constant
do not need an isothermal
- Allows for high throughput diagnosis of parasitic diseases in large scale studies
- Bead-based flow cytometry assay that allows for simultaneous detection of different targets (parasite species or genotypes) in the same reaction using very low volumes
- Very useful in parasite genetic diversity and drug resistant allele studies of your parasites.
- DNA is isolated and undergo amplification particularly specific gene fragment afterwards and subject in luminex system with the use of color beads
- This multiplex assay will be used of these beads in a single well and with the use of biotinylated primer can capture it in a fragment then add streptavidin or streptavidin pico urethane to detect the presence of the organism.
- ANalysis is normally done using this laser excitation green and red for luminex xmap technology.
LUMINEX XMAP TECHNOLOGY
Conventional and Real-time PCR or Conventional PCR
o Cryptosporidium spp.
o Cylospora cayetanensis
o Entamoeba histolytica
o Entamoeba dispar
Conventional and Real-time PCR
Conventional and Real-time PCR or Conventional PCR
o Giardia duodenalis
o Microsporidia
Conventional PCR
where amplification products of conventional PCR is loaded
Anong gel
Agarose gel
- measures the fluorescence signal in the reaction tube per cycle and is proportional to the amount of accumulated amplified product (check for the standard curve and fluorescence signal in the reaction per cycle depending on the cycle made ( take note that is it proportional to the accumulated amplified product)
- Increase of no. amplified product there increases intensity fluorescence signal.
anong PCR
real time PCR
Differentiates Cryptosporidium hominis from Cryptosporidium parvum.
anong PCR
TaqMan-based real-time PCR
What does the Generic TaqMan assay targets to detect Cryptosporidium species?
18S rRNA
to identify C. hominis and C. parvum or differentiate them through this assay
anong type na taq man assay
Two species-specific TaqMan assays
- Detect the pathogenic E. histolytica and non-pathogenic E. dispar.
- simultaneously detect E. histolytica, Giardia intestinalis and Cryptosporidium spp. in one tube using parasite-specific probes
- Uses two species-specific probes encompassing new SSU RNA regions of the ribosomal DNA-containing episome
A. Single-tube multiprobe real-time PCR assay
B. Multiplex real-time PCR
ABA
primers and TaqMan probes were designed on a small subunit ribosomal RNA gene to detect what two organisms?
E. histolytica and Giardia duodenalis
To detect Cryptosporidium spp., it is directed to?
Cryptosporidium oocyst wall protein
(COWP)
To detect Schistosoma spp., it is directly against what gene?
cytochrome C oxidase gene
To differentiate Taenia spp OVA
familiarize urself
- PCR restriction fragment length polymorphism (PCR-RFLP)
- Multiplex PCR targeting mitochondrial DNA
- Nested PCR targeting – Tso31 gene encoding the
- T. solium oncosphere specific protein
- LAMP technology - targets COX 1 and cathepsin L-like cysteine peptidase (clp) genes
Pls study the PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM PROTOCOL FOR ASCARIS SPP
Thank u
Play 1:16:20 to be guided, sandali lang siya
wag tamarin https://youtu.be/ewqWab23M08?si=29iyJOMTIldvsxtx
- Use antibodies (monoclonal or polyclonal) to detect parasite antigens in blood, stool, urine or other body fluids
- Great potential in improving diagnostic accuracy of parasitic infections in field settings that still rely on the microscope
- Without the need for :
o Electricity
o Sophisticated equipment
o Extensive training of laboratory personnel - Employ immunochromatographic methods
- Results are available within 15 MINUTES (Advantage) within 20mins release the result if positive.
RAPID DIAGNOSTIC TEST
RDT
Principle, except:
A. Lysing agent labeled Ab
B. Nitrocellulose strip
C. Bound Ab/Ag
D. Free labeled Ab
E. Cellulose
E
RDT
Interpret Results
- control, + test band
- control, + test band
- control, - test band
- control, - test band
- Positive
- Invalid
- Negative
- Invalid
RAPID DIAGNOSTIC TEST
TOF. As the level of the parasite increase the intensity of the band being form in the control will be decrease.
T
- Is a lateral flow immunochromatographic device that detects protein [antigen (Ag)] derived from the blood stage of malaria parasites.
- Volume : 5 to 20 μL
- Lysed to release the Ag from within red blood cells and parasites from within these cells (a variable amount of Ag is also present in the serum)
- Test produces a series of visible lines to signal the presence or absence of Ag in the blood sample
RDTS FOR MALARIA
Pls study the diagram for the RDTs for Malaria
thx
RDTs for Malaria
The end product after the action of the labeled antibody and bound antibody
color
Red
RDTs for malaria detect either?
P. falciparum histidine rich protein 2 (PfHRP-2) or parasite lactate dehydrogenase (pLDH)
RDTS FOR MALARIA
Good levels of sensitivity have been achieved with what levels of parasitemia?
interpret
> 100 parasites/µL blood
error
skip
RDTS FOR MALARIA
sensivity drops in the presence of parisatemia
anong level
<100 parasites/uL
- HRP - 2 /pLDH (Pf/Pan) Combo Test
- Has different kit for detection (pero isa lang nadedetect na specific organism)
▪ Falciparum
▪ Vivax
▪ Ovale
▪ Malariae
The Care StartTM Malaria
- a three-band RDT that can detect both PfHRP - 2 (P. falciparum histidine rich protein-2) and pan-pLDH from infected blood
(Pf/Pan) Combo Test
(Pf/Pan) Combo Test
- The presence of an HRP - 2 line indicates infection with?
- P . falciparum , and the presence of a pan-pLDH line indicates infection with?
*P . falciparum
* one or more of the non - P . falciparum species
- Detect taeniasis caused by the adult worm of the cestode Taenia solium
- It is a POCT test a control line and test line where you put your sample
- Gold colloid particles
MAGNETIC IMMUNOCHROMATOGRAPHIC TEST (MICT)
MAGNETIC IMMUNOCHROMATOGRAPHIC TEST (MICT)
- neurocysticercosis caused by the larval forms has been developed based on two specific T. solium excretory - secretory proteins
- These two ES protein detects antibodies against taenia solium and use as point of care case or confirmation the immunochromatographic test.
- ES33 - Excretory Secretory Protein 33
- ES38 - Excretory Secretory Protein 38
- Use to detect schistosoma spp
- Eliminates the need for a fecal sample which is more difficult to collect from patients.
- A useful supplement to Kato-Katz examination for the rapid detection of intestinal schistosomiasis
CIRCULATING CATHODIC ANTIGEN (CCA)
CIRCULATING CATHODIC ANTIGEN (CCA)
TOF. A negative association between increasing intensity of CCA urine-dipstick test band and fecal egg count was observed.
F (posi)
- To detect Leishmania antibodies revolutionized the diagnosis of visceral leishmaniasis.
RK39 ANTIGEN AND RK38 POLYPROTEIN-BASED
- ONLY detect Leishmania spp in INDIAN continent but in the part of african subcontinent hindi kaya idetect mababa yung specificity rate.
- can detect AFRICAN continent belongs doon were yung Leishmania prevalent
identify each antigen
- RrK39 antigen
- rK38
