Parasitic and Fungal diseases and Serologic test

Parasites

microorganisms that survive by living off of other organisms referred to as your host.

Protozoa

single-celled organisms that live and multiply inside human hosts. These are microscopic and can cause diseases like your malaria or amebiasis

Helminths

multicelled organisms living alone or in humans. These are your worms like your tapeworms or your round worms.

Ectoparasites

multicelled organisms that live on the skin. Think of these as your lies, fleas or mites.

Neglected Tropical Diseases (NTDs)

are major parasitic diseases causing morbidity and mortality worldwide.

TRUE

TRUE OR FALSE: Bacteria are unicellular with have simple life cycles and limited epitope variation. Parasites, they are multicellular, elicit more complex immune responses. They can change forms, live in different tissues, and even alter how they look to the immune system

Heterogeneity and Antigenic expressions

is a key feature of a parasitic agents

Antigenic concealment

• Parasites hide their antigens from the host.

• It can be by remaining inside of the host’s cell without their antigens being displayed. 

• Must have a specific target cell.

• Develop strategy to enter and survive in host cell.

Antigenic variation

Parasites change their surface antigen so they would not be recognized by your immune system.

• Random mutation

• Genetic recombination

• Gene switching

Three main mechanisms of antigenic variation:-

Gene switching

• Most dramatic form of antigenic variation.

• Organisms may carry upwards of one thousand different genes, allowing expressions of distinct surface molecules.

• Parasite switches from one gene to another, persisting while the immune system tries to catch up.

Genetic recombination

• Rearrangement of genes within the organism allows development and expression of new epitopes on the parasite surface.

• These epitopes are unrecognized by the previous immune response.

Random mutation

• Parasite generates novel antigens by random mutation

• Some evolve mechanisms allowing sufficient frequency to evade the immune system continuously.

Antigenic shedding

• Similar to bacteria that shed capsular material

• Although antibody is formed against the parasite, the antibody attaches to the shed antigen rather than the parasite.

• Allows the parasite to escape the immune response.

Antigenic mimicry

• Occurs when the parasite expresses epitopes similar or identical to host molecules. 

• Protects the invading parasite from being recognized and eliminated by the immune system.

• Cause host cross-reactivity, leading to autoimmunity

Immunologic subversion

This is achieved by avoiding the effector mechanisms of the immune response. The weapons our body uses to fight back.

Immunologic diversion

• Occurs when the parasite causes the immune system to produce proteins that divert its attention.

• Cause an increase in production of beta interferon (IFN-β), allowing for increased parasite survival

TRUE

TRUE OR FALSE: When demonstration of the parasite in biological or tissue samples is not possible, serological testing is the gold standard for diagnosis.

• Tests that detect parasitic antigens

• Tests that detect antibodies against the parasite

Serological-based assays are divided into:

• Plasmodium falciparum

• Plasmodium malariae

• Plasmodium ovale

• Plasmodium vivax

Causative agent of malaria

bite of Anopheles mosquitoes

Malaria is a bloodborne parasite and is transmitted through the?

• fever

• anemia

• splenomegaly

Malaria is characterized clinically by?

1. Shaking chills

2. Fever (up to 40 deg Celsius or higher)

3. Generalized diaphoresis, followed by resolution of fever

Malarial attack (paroxysm) in order:

6-10 hours

Duration of malarial attack

synchronous rupture of erythrocytes

The cause of malarial attack is initiated by the?

RTS,S/AS01

directed against the deadly P. falciparum strain.

FMP2.1/AS02

recombinant protein based on apical membrane antigen 1 from the 3D7 strain of P. falciparum

thick and thin blood films

Laboratory evaluation for suspected malaria relies on timely examination of?

Microscopic Examination

gold standard for laboratory confirmation of malaria.

Immunocapture Assays

detects Plasmodium-specific antigens and provide a high degree of sensitivity and specificity for diagnosing falciparum malaria.

• Lactate dehydrogenase

• Histidine-rich protein II

What antigens are detected in immunocapture assays?

Immunofluorescence Antibody Testing (IFA)

• has been a reliable serologic test for malaria in recent decades.

• Though time-consuming and subjective, it is highly sensitive and specific

• Previously used for detecting Plasmodium-specific antibodies in blood bank units to prevent transfusion-transmitted malaria.

1. Epidemiological surveys

2. Screening potential blood donors

3. Providing evidence of recent infection in non-immune individuals

Uses and applications of IFA:

Enzyme Immunoassay (EIA)

a rapid diagnostic test has been developed to differentiate Plasmodium falciparum from less virulent malaria parasites.

BinaxNOW Malaria Test

• Lateral flow immunochromatographic assay

• Targets the histidine-rich protein (HRPII) specific to P. falciparum (P.f.) and a panmalarial antigen common to all four malaria species.

Polymerase Chain Reaction (PCR)

• Detects parasite nucleic acids for diagnosis of malaria.

• More sensitive than blood smear microscopy, but has limited utility for acutely ill patients in standard healthcare settings.

• More useful for confirming the species of Plasmodium after diagnosis is established by smear microscopy or rapid diagnostic tests (RDTs).

Entamoeba histolytica

pathogenic species from the genus Entamoeba, characterized by chromatin on the nice membrane.

Amoebic dysentery

blood diarrhea with abdominal cramps; may lead to ulceration, perforation, and peritonitis.

Amoebic colitis

milder; may mimic ulcerative colitis with nonbloody diarrhea, constipation and weight loss.

Amoebic liver abscess

most common extraintestinal form; presents with fever and right upper quadrant pain.

Microscopic examination of fecal specimens

usual method for identifying Entamoeba; it is easier and provides more definitive results that serum antibody testing

Enzyme immunoassay (EIA)

• available for Entamoeba identification, using enzyme-conjugated antigen or antibody, depending on the assay.

• Detectable antibody titers may persist for months or years after successful treatment. A positive result doesn’t always mean there’s an active infection.

• Useful for extraintestinal diseases

1. 95% of extraintestinal cases

2. 70% of intestinal cases

3. 10% of asymptomatic cyst passers

EIA detects antibodies in:

Real-time PCR (TaqMan assay)

• techniques can identify Entamoeba species; specificity equals microscopy, but sensitivity is higher.

• Targets 18S rNA gene 

• Can detect both E. histolytica and E. dispar in one reaction

TRUE

TRUE OR FALSE: Adult male and female blood flukes inhabit the veins of the mesentery or bladder.

granuloma formation

Chronic infection of schistosomiasis leads to?

reducing antigen expression

Adult schistosomes evade immune defenses by?

glycolipid and glycoprotein coat

Schistosomes enclose themselves in a?

detecting eggs in feces or urine (direct wet mount or formalin-ethyl acetate concentration methods)

Gold standard of schistosomiasis

Falcon Assay Screening Test-ELISA (FAST-ELISA)

• It is a serological test that detects antibodies against the parasite, often using microsomal antigens from Schistosoma mansoni.

• It is typically used as a screening tool, with positive results then often confirmed using another method like the enzyme-linked immunoelectrotransfer blot (EITB).

Toxoplasma gondii

is an ubiquitous protozoan parasite that infects humans by ingestion of infective cysts (oocysts).

Congenital toxoplasmosis

Occurs when a woman becomes infected just before or during pregnancy.

TRUE

TRUE OR FALSE: In toxoplasmosis, if infection occurs before pregnancy, maternal antibodies protect the fetus. If the infection occurs again during pregnancy, the parasite can cross the placenta, putting the fetus at serious risk for complications.

• Miscarriage, stillbirth

• hydrocephalus

• blindness (chorioretinitis)

Possible outcomes of congenital toxoplasmosis:

TRUE

TRUE OR FALSE: T. gondii can prevent the fusion of lysosomes with phagosomes, hence can survive indefinitely.

Sabin-Feldman Dye Test

• Highly specific and sensitive test for diagnosing toxoplasmosis. Detects IgG antibodies in the patient’s serum.

• Measures the ability of antibodies to neutralize live parasites.

• Toxoplasma gondii considered the reference (gold standard) for toxoplasmosis serodiagnosis.

• Requires live parasites, limiting its routine use

Based on the detection of Toxoplasma-specific antibodies in a patient’s serum.

Principle of Sabin-Feldman Dye Test

possible recent infection or false positive IgM

In Toxoplasmosis, if IgM and IgG is both positive, what does it indicate?

acute infection/false positive IgM result

In Toxoplasmosis, if IgG (-), IgM (+), what does it indicate?

past infection

In Toxoplasmosis, if IgG (+), IgM (-), what does it indicate?

IgG Avidity Test

• When both IgM and IgG antibodies are detected in a pregnant patient

• Determines whether IgG antibodies are from a recent or past infection.

Fungi

eukaryotic organisms that are spore-bearing, have absorptive nutrition, lack chlorophyll, and reproduce sexually and asexually

Yeast

unicellular organisms that reproduce by budding.

Mycelial

multicellular organisms that reproduce through spores or conidia.

Mycoses

• Infections caused by fungi.

• Can cause hypersensitivity, mycotoxicosis, or tissue infections.

Systematic

• Fungal infections deep within the body; can involve the deep viscera and spread throughout the body.

• Originate in the lungs and then disseminate to other locations.

Cutaneous

• Fungal infectious beneath the skin caused by saprophytic fungi that live in soil and on vegetation.

• Occurs by direct implantation of spores or mycelial fragments into a puncture wound in the skin.

Superficial

• Fungal infections that involve the keratinized body areas and rarely invade deeper tissues.

• Produce itchiness and cracking of the skin.

Subcutaneous

• Fungal infections that are restricted to the outer layers of the skin.

• Cosmetic in nature and are not life threatening.

Opportunistic

Generally harmless in its normal habitat but can become pathogenic in an immunocompromised host.

Innate immune response

serves as the first line of defense. The skin and mucous membranes act as physical barriers to prevent fungal entry.

Adaptive immune response

cell mediated immunity involving T cells and cytokine production plays the most important role in controlling fungal infections.

Pattern Recognition Receptors (PRRs)

special receptors found on immune cells that detect unique structures on pathogens called as PAMPS or pathogen associated molecular patterns.

Dectin-1

recognizes β-1,3 glucan (major components of your fungal cell walls)

Dectin-2

recognizes α-mannans (another key fungal carbohydrate)

Mannose receptors

recognize exposed mannose residues

Cryptococcosis

is a systemic mycosis caused by the Cryptococcus species complex.

Cryptococcus neoformans

a saprophyte with worldwide distribution; source of infection is aged, dried pigeon droppings.

Cryptococcus gattii

found in tropical and subtropical regions, especially near eucalyptus trees; mainly in northern Australia and Papua New Guinea.

Polysaccharide capsule

Main factor responsible for pathogenicity. The reason why Cryptococcus can survive and cause disease even in an active immune system.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS)

Used to rapidly discriminate C. neoformans and C. gattii (This allows us to rapidly differentiate them.)

Latex Agglutination (LA) Tests

• for detection of polysaccharide antigen of the C. neoformans species complex in CSF and serum. 

• Serum antigen is more sensitive than CSF testing in HIV-infected patients. 

• Titration of positive results is used to assess prognosis and establish baseline for monitoring treatment response

1:2

What titer:

suggests possible infection (may occur without clinical disease)

1:4

What titer:

indicates active infection

Lateral Flow Immunochromatographic Assay (LFA)

• Detects cryptococcal antigen in <15 mins

• Rapid, highly sensitive, and specific method for diagnosis. It is very crucial especially in critical ill or immunocompromised patients.

Indirect Immunofluorescence Assay (IFA)

• Detects antibodies to Cryptococcus neoformans

• Useful when antigen tests are negative

• Positive result indicates a current or recent infection

• Can be combined with antigen testing to assess prognosis.

• Antigen may remain detectable for months after successful treatment

Histoplasma capsulatum

a facultative intracellular pathogen

Complement Fixation (CF) Test

• Detects antibodies

• Cross-reactions may occur (aspergillosis, blastomycosis, coccidioidomycosis), but with lower titers.

Immunodiffusion (ID) Test

Detects H and M antibodies to fungal glycoproteins

Coccidioidomycosis

Acquired through inhalation of soil or dust containing arthrospores of Coccidioides immitis. It is tiny and easily airborne.

Primary pulmonary coccidioidomycosis

Most common form of coccidioidomycosis, usually affecting the lungs

Primary cutaneous coccidioidomycosis

very rare, happens when spores directly enter through the skin

Disseminated coccidioidomycosis

more serious form of coccidioidomycosis

Direct Microscopic Methods

• Calcofluor white stain of respiratory specimens (to highlight fungal elements under fluorescence microscopy)

• Histopathologic examination of tissue samples

Enzyme Immunoassays (EIA)

• Most common

• Allows the specific detection of IgM or IgG antibodies.

• A positive result is a highly sensitive indicator for coccidioidal infection

Hypersensensitivity Testing

Performed using intradermal injections as screening method (injection of antigens to check for delayed type hypersensitivity reaction)

Tube Precipitin Test (TPT)

• One of the original methods for detecting C. immitis infection

• Detects IgM antibodies against C. immitis

Complement Fixation (CF) Test

• Most widely used quantitative serodiagnostic test for C. immitis infection

• Detects IgG antibodies (often called the “IgG test”)

Aspergillosis

Most common life-threatening opportunistic invasive mycosis in immunosuppressed patients

Invasive Pulmonary Aspergillosis (IPA)

occurs in neutropenic immunosuppressed patients