INTESTINAL NEMATODE INFECTIONS 1. Used to increase diagnostic sensitivity of microscopy; labor-intensive have poor reproducibility owing to operator error, and usually require centrifugation steps 2. Enables assessing the prevalence and intensity of infection in nematode control programmes identify techniques

1. Floatation and conc techniques 2. Kato katz

(F) Advances of DX Parasitology: (lecture-based) Flashcards by ASHLEY JARED DEFENSOR

Gold standard
Allows detection of parasites
Morphologically distinct parasites are differentiated
Requires high parasite density to achieve a good level of sensitivity

MICROSCOPY

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Enumerate the Basic Microscopic Techniques (5)

DFS
Kato Katz
Formol-Ether-Sedimentation Technique
Harada-Mori
Mcmaster technique

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TOF. For a good level of sensitivity, there should be a low parasite densitive.

F (high parasite density)

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2 Reagents used for DFS

Lugol’s iodine and NSS

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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)

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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)

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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

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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

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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

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FEST

Enumerate the 4 layers in order

upper to last layer

Ether, Fecal debri, Formol Water, Sediment (containing the parasites)

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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

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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

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harada mori

identify
* Incubation days
* Centrifuge (RPM for how many minutes)

3-7; pero p’wede rin 10 days
1500rpm for 2 minutes

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Harada Mori

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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

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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

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Power Objective

Kato Katz
FEST
Harada-mori
Mcmaster technique

A. 10x
B. 40x
C. both
D. neither

may repeating answers

CCAA

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INTESTINAL NEMATODE INFECTIONS

Microscopic techniques used for diagnosis

DFS and kato-katz

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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

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INTESTINAL NEMATODE INFECTIONS

TOF. Larvae of Ascaris lumbricoides may be observed in the sputum suring pulmonary migration.

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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)

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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.

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INTESTINAL NEMATODE INFECTIONS

Familiariza yourself with the advantages and disadvantages

pati na rin intestinal tapeworm infections + liver lung and blood flukes infection advantages and disad

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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)

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# 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.

# 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.

Microscopic techniques used for diagnosis 1. Kato-Katz Method 2. 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

Microscopic techniques used for diagnosis 1. Wet mount preparations of fresh or concentrated stool 2. Preianal scraping (Graham’s test) 3. 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

Microscopic techniques used for diagnosis 1. Wet mount preparation of stool/ bile/ sputum/ urine after the concentration by sedimentation or flotation techniques 2. Membrane filtration technique using nucleopore filters for schistosomal infections 3. Kato-Katz method ○ Sedimentation or flotation techniques A. INTESTINAL NEMATODE INFECTIONS B. INTESTINAL TAPEWORM INFECTIONS C. LIVER,LUNG AND BLOOD FLUKES INFECTION

* 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.

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

Pls study the ANTIBODY DETECTION TEST OFFERED AT CDC

okay thx ## Footnote 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

# 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

# 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

* 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

* 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

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 1. separate double stranded DNA at high temp 94-95°C 2. 50-60°C ,facilitates binding of primer to its complementary sequence 3. 72-74°C DNA synthesis process | identify each step ## Footnote 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 1. 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. 2. detect multiple species of parasite in a single sample in a single tube 3. 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

* 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) ## Footnote + luminex-based technologies

# LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) TOF. Do not require extraction of parasite DNA (as compare to conventional PCR).

# 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.

# LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) Measurement A. Naked eye B. Real-time C. Both D. NOTA

# LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP) Temperature

60-65C constant ## Footnote 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

1. Detect the pathogenic E. histolytica and non-pathogenic E. dispar. 2. simultaneously detect E. histolytica, Giardia intestinalis and Cryptosporidium spp. in one tube using parasite-specific probes 3. 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 ## Footnote 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

# RDT Interpret Results 1. + control, + test band 2. - control, + test band 3. + control, - test band 4. - control, - test band

1. Positive 2. Invalid 3. Negative 4. 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.

* 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

(F) Advances of DX Parasitology: (lecture-based) Flashcards

Rupert-based