IS LAB MIDTERMS BACTERIAL DISEASES

MODULE 4: DISEASES AND THEIR SEROLOGIC TESTS

UNIT 1: Bacterial Diseases and their Serologic Tests Causative Agent Laboratory Diagnosis

1. Syphilis Treponema 1) Direct detection of spirochetes

“Tags” (opsonins) facilitate recognition by macrophages, and so the organism is
pallidum -Darkfield
destroyed. Intracellular bacteria, on the other hand, are eliminated by promoting the
-Fluorescent Ab
natural lytic machinery in the infected cell. CD4+ T-cell recognition of cell surface changes
results in secretion of molecules (cytokines) which enhance destruction of the 2) Nontreponemal serological tests
intracellular organisms. -VDRL
-RPR
Extracellular bacteria Intracellular bacteria -TRUST
Phagocytosis, the primary means of Key cells in immunity to intracellular
eliminating extracellular bacteria, is bacteria are cytokine activated 3) Treponemal serological tests
enhanced by molecules generated macrophages. The cytokines involved are -FTA-ABS
following the activation of B cells, T cells, derived from activated natural killer cells -EIA
and complement. Nonspecific recognition and CD4+ T cells. -MHA-TP or Serodia TP-PA
of bacteria by phagocytes is termed direct -DNA probe
phagocytosis, while phagocytosis that 2. Typhoid fever Salmonella 1) Typhidot
follows the binding of opsonin is termed typhi G+
opsonin-mediated phagocytosis. 2) Widal test

3. Streptococcal Streptococci G+ 1) Streptococcal Ag

infection a. Culture
b. Enzyme Immunoassay or Latex Agglutination
c. Molecular Methods
d. Serotyping

2) Streptococcal Ab
a. Antistreptolysin O (ASO) Testing
b. Anti-DNase B Testing
c. Streptozyme Testing
4. Rickettsial Rickettsiae G- Serodiagnosis: method of choice
infection
IFA, micro-IF: gold standard

IPA, ELISA, IBA

5. Mycoplasma Mycoplasma Detection of Ab
infection pneumoniae - EIA
-particle agglutination
-IFA
-complement fixation
 NATURE OF IMMUNE RESPONSE
SYPHILIS • T cells and macrophages play a key role in the immune response.
• Primary lesions- presence of both CD4+ and CD8+ T cells, which produce cytokines that
CAUSATIVE AGENT MODE OF activate macrophages, leading to phagocytosis and healing the primary chancre.
TRANSMISSION • T. pallidum is capable of coating itself with host proteins, which delays the immune
• Treponema pallidum sub pallidum Primary: system’s recognition of the pathogen.
• No natural reservoir in environment sexual contact • The rare treponemal proteins, or TROMPS, are important in bringing about complement
• must multiply within a living host (abraded skin or activation that ultimately kills the organism.
T. pallidum Agent of mucous membranes) • In treponemes, 2 classes of Ag:
subspecies (1) Ag restricted to one or a few species
pertenue Yaws: tropics Congenital infection
(2) Ag shared by many different spirochetes
endemicum Nonvenereal endemic syphilis or bejel: Parenteral exposure
• Specific antibodies against T. pallidum and nonspecific antibodies against the protein
desert
NOTE: Pathologic antigen group common to pathogenic spirochetes are formed.
carateum Pinta: Central and South America
treponemes are • Specific treponemal antibodies in early or untreated early latent syphilis are
rapidly destroyed by predominantly IgM. Then this is followed by the appearance of IgG, which have greatest
• outer membrane: phospholipid bilayer with very few heat, cold, and drying elevation seen in secondary syphilis.
exposed proteins, including treponemal rare outer membrane out, thus they are • Nontreponemal Ab: reagin Ab- produced by infected px against components of their
proteins (TROMPs) almost always spread own. These Ab are also produced by px with -
• scarcity of such proteins delays the host immune response by direct contact. other infectious diseases:
measles, chickenpox, hepatitis, infectious mononucleosis, leprosy, tuberculosis,
STAGES OF SYHILIS leptospirosis, malaria, rickettsial disease, trypanosomiasis, lymphogranuloma venereum
Incubation Primary Secondary Latent Relapses 2’ Tertiary noninfectious conditions:
3 weeks painless rash -body “hidden” 20-30% “late” autoimmune disorders, drug addiction, old age, pregnancy, recent immunization
10-90 chancre no Sx reexperience most
days 28-42 days 2-8 weeks 1 yr/ 5-20yr 2’ Sx destructive LABORATORY DIAGNOSIS

heals w/o heals w/o can occur 1 yr after

treatment scarring several infection/
2-12 weeks times any time

open sores miscarriage, px may never

pus: stillbirth, experience
condyloma congenital this stage
lata syphilis gummata,
large sores,
nervous cardiovascular 3 Main Types of Test
system abn No syphilis, 1) Direct detection of spirochetes
occurrence: neurosyphilis 2) Nontreponemal serological tests
highly highly Not 3) Treponemal serological tests
contagious contagious contagious contagious
1) Direct detection of spirochetes II. Nontreponemal Serological Tests
Test Darkfield Fluorescent Ab Test Venereal Disease Research Laboratory Rapid Plasma Reagin
Ag T. pallidum from px T. pallidum from px (VDRL) Test (RPR) Test
Ab None Antitreponemal Ab fluorescent tag both a qualitative & quantitative slide modified VDRL test involving
Descr keep all incident light out of the field performed by either: flocculation test for serum macroscopic agglutination
except for that captured by the direct method- fluorescent-labeled modification for use on CSF
organisms themselves Ab conjugate to T. pallidum Ag suspension (alcoholic solution) cardiolipin-containing Ag suspension
0.03% cardiolipin is bound to charcoal particles,
Pathogenic treponemes: indirect method- Ab specific for T. 0.9% cholesterol makes test easier to read
corkscrew morphology pallidum and a second labeled anti- 0.21% lecithin
flexing motility immunoglobulin antibody serum specimens-heated at 56˚C for Ag is similar to the VDRL Ag w/ the
30 minutes to inactivate complement addition of EDTA, thimerosal, and
Specimen: serous fluid from a lesion choline chloride, which stabilize the
antigen and inactivate complement
Negative test does not exclude a so that serum does not have to be
diagnosis of syphilis. heat inactivated before use
Cmt Requires active lesion Requires active lesion
must have good specimen specimen does not have to be live III. Treponemal Serological Tests
experienced technologist inexpensive more specific than darkfield Test Fluorescent Treponemal Antibody Agglutination Enzyme Polymerase
Absorption Tests Immunoassay Chain Reaction
(FTA-ABS) Test (EIA) (PCR)
SEROLOGICAL TESTS FOR SYPHILIS indirect fluorescent Ab test detect specific reagin II, uses isolating &
Nontreponemal Treponemal treponemal Ab a cardiolipin amplifying a
check for the presence of Ab referred to detect Ab that are directed against the T. most used confirmatory tests Ag similar to sequence of
that used in DNA that is
as reagin, that are formed against pallidum organism or against specific dilution of heat-inactivated px serum Serodia TP-PA the VDRL test unique to a
cardiolipin, a lipid released from damaged treponemal antigens incubated w/ sorbent consisting extract (particle particular Ag
host cells (IgG / IgM) of nonpathogenic treponemes (Reiter agglutination capture a
1-4 weeks after the appearance of the become positive earlier than strain) test): specific class currently still
use colored of Ab either being studied
primary chancre, nontreponemal tests nontreponemal tests Slides: Nichols strain of T. pallidum gelatin particles IgM or IgG
generally become positive coated w/
based on flocculation, a specific type of more difficult to perform, time consuming Px Ab + binds to T. pallidum Ag treponemal Ag
precipitation that occurs over a narrow used traditionally for confirmation rather
Intensity of Green color reported 0-4+ -more sensitive in
range of antigen concentrations than screening (-): no fluorescence detecting primary
(>2+): reactive syphilis
-13-14% px appear nonreactive during primary stage
-Reagin Ab titers peak during the secondary and early latent stages, hence, almost all
patients show reactive results in these stages.
-Few patients may appear nonreactive during the early stage of primary syphilis, but 100%
of tests almost always show reactive results during the secondary and latent stages.
-Patients who show reactive results in treponemal tests remain so for the rest of their
lives.
TYPHOID FEVER STREPTOCOCCAL INFECTION

CAUSATIVE AGENT CLINICAL MANIFESTATIONS CAUSATIVE AGENT CLINICAL MANIFESTATIONS

• Salmonella typhi G+ bacilli family • mild or severe, may include prolonged • Streptococci G+, catalase -, pair of chain, • 2 most common sites of infections:
Enterobacteriaceae fever, severe headache, malaise, spherical, ovoid, or lancet-shaped upper respiratory tract and the skin, with
• intestinal pathogen constipation or diarrhea, rose-colored • outermost cell wall component contains pharyngitis and impetigo being the most
• Paratyphoid fever (milder): caused by spots on the trunk and an enlarged spleen two major proteins known as M and T, common clinical manifestations
Salmonella paratyphi A or B • start of symptoms and these determine the serogroup or • Severe invasive infections with group A
• MOT: feces or urine -ingestion -8-14 days typhoid fever serotype. The serotype is based on minor streptococci have also been associated
Contaminated H2O and food: most -1-10 days paratyphoid fever variations in the proteins that can be with toxic shock syndrome and
common sources of infection. • Px infected with Salmonella can become identified serologically. necrotizing fasciitis.
• Mary Mallon: 1st asymptomatic carrier chronic carriers of the bacteria. • Interior to the protein layer is the • 2 main damaging sequelae to group A
of Salmonella typhi “Typhoid Mary” group-specific carbohydrate that divides streptococcal infections: acute rheumatic
streptococci into 20 defined groups fever, poststreptococcal
LABORATORY DIAGNOSIS designated A through H and K through V, glomerulonephritis
known as the Lancefield groups.
3 Ag that is of importance in serologic testing: Streptococcus pyogenes, which belongs to 1) Acute rheumatic fever
O antigen H antigen K antigen (capsular)/ Lancefield group A, is one of the most ➢ Sequela to pharyngitis or tonsillitis in 2-
common and ubiquitous pathogenic 3% of infected individuals, but not from
(cell wall) (flagellar) Vi antigen (virulence)
bacteria and causes variety of infections. skin infections.
outer polysaccharides of 2 forms: phases 1 & 2 antiphagocytic
cell wall Only 1 H proteins is important virulence factor
• M protein: major virulence factor of the ➢ Most likely due to Ab or cell-mediated
group. And immunity to Group A immunity originally produced against
used to subdivide the synthesized at any one for S. typhi
streptococci appears to be associated streptococcal antigens but that cross-
salmonellae into group A-I time, depending on which used for serotyping of S.
with antibodies to the M protein. react with human heart tissue. Chief
gene sequence is in the typhi
• Other virulence factors are exoantigens among the Ab is those directed toward
correct alignment for
or exotoxins, including pyrogenic the M proteins, which have at least three
transcription into mRNA
exotoxins A, B, and C. epitopes that resemble antigens in heart
• Antibodies are produced to the tissue, permitting cross-reactivity to
Typhidot Widal test following exoantigens: enzymes occur.
enzyme immunoassay that detects IgM • direct agglutination test: used to detect streptolysin O, deoxyribonuclease B
and IgG Ab against the outer membrane a rise in the Ab titers against S. typhi Ag in (DNase B), hyaluronidase, nicotinamide 2) Poststreptococcal glomerulonephritis
protein of Salmonella typhi px serum adenine dinucleotidase (NADase), and
• Anti-O and anti-H Ab rise early at 1-2 ➢ Sequela of either infection of the skin
streptokinase. or the pharynx
weeks after infection, peak at 3-5 weeks • Culture and rapid screening methods
• Anti-A and anti-B Ab (against Vi Ag) rise ➢ deposition of Ab-streptococcal Ag
are routinely used for diagnostic testing
2-3 weeks after infection and persist for immune complexes in the glomeruli
early in the infection. However, diagnosis
4-5 weeks of the sequelae such as
• Significant titers glomerulonephritis and acute rheumatic
- 80 for unvaccinated patients fever is best achieved by detection of
-160 for vaccinated patients antibodies.
DETECTION OF GROUP A STREPTOCOCCAL ANTIGENS
Culture Enzyme Molecular Methods Serotyping RICKETTSIAL INFECTION
Immunoassay
or Latex CAUSATIVE AGENT CLINICAL MANIFESTATIONS
Agglutination • ickettsiae: short rods, • Rocky Mountain spotted fever (RMSF) caused by R.
sheep blood agar: small throat swabs hybridization of specific Identification coccobacilli, obligate, rickettsii, sx occur 7 days after tick bite: fever, severe
translucent colonies w/ clear within 2-30 min rRNA sequences and of M protein intracellular, G- headache, malaise, myalgia, nausea, vomiting, abdominal
zone beta hemolysis real-time PCR antigens by
• 2 groups: spotted fever pain, cough, rash (hands and feet, proceeds to trunk,
High specificity Genotyping techniques precipitation
Presumptive identification: but with varying involving PCR with type- group (SFG), typhus group appears in 3-5 days after the beginning of other sx)
susceptibility to bacitracin, sensitivity amplification of a specific (TG) • infects endothelial cells= increase in vascular
PYR activity, growth in the depending on portion of the emm antisera • diseases are associated permeability and focal hemorrhages
presence of trimethoprim- commercial kits gene, which codes for with arthropods, ticks, • Murine typhus, caused by R. typhi
sulfamethoxazole the M protein mites, lice, fleas Sx: cough and chest infiltrate suggestive of pneumonia
• Humans: accidental Rash: one-half of patients
DETECTION OF STREPTOCOCCAL ANTIBODIES hosts except for Rickettsiia severe manifestations: seizures, coma, respiratory failure
Antistreptolysin O (ASO) Anti-DNase B Testing Streptozyme Testing prowazekii
Testing
•Presence of Ab to •useful in px suspected of • slide agglutination SEROLOGIC DIAGNOSIS
streptolysin O = recent glomerulonephritis preceded by detection of Ab to
• Serodiagnosis: method of choice
streptococcal infection in px streptococcal skin infections, ASO streptococcal Ag
suspected of acute rheumatic Ab not stimulated by this type of • Sheep red blood • Weil-Felix test: agglutination test, used to detect Ab in px with rickettsial infections
fever or poststreptococcal disease cells: coated with based on cross-reactivity of the px Ab with polysaccharide Ag present on OX-9 and OX-2
glomerulonephritis ff a throat • Ab to DNase B may be detected streptolysin, strains of Proteus vulgaris and the OX-K strain of Proteus mirabilis.
infection in patients with acute rheumatic streptokinase, • Current serologic assays are organism-specific:
• classic hemolytic 1st method fever who have a negative ASO hyaluronidase, DNase, -indirect fluorescent assays (IFA)
for determining the ASO titer test result. and NADase so that -microimmunofluorescent assays (micro-IF)
• Ab in the px serum • test based on a neutralization Ab to any of the
-immunoperoxidase assays (IPA)
neutralize hemolytic activity method streptococcal Ag can
of streptolysin O • anti-DNase B Ab present: be detected. -ELISA
• enough Ab present, neutralize rgt DNase B, • Hemagglutination: -immunoblot assays (IBA).
streptolysin O is neutralized, preventing it from positive, Ab to 1 or • IFA and IPA require the whole bacterium as the reagent, while ELISA and IBA use
no hemolysis occurs depolymerizing DNA more of these Ag are rickettsial antigens adsorbed onto a solid phase or nitrocellulose membrane.
• titer expressed in either • DNase is measured by its effect present • IFA test , micro-IF : gold standard for detecting rickettsial Ab
Todd units (streptolysin rgt on a DNA methyl-green • excellent screening
std is used) or in international conjugate. This complex is green tool, used in
units in its intact form, but when conjunction with the
•single ASO titer: hydrolyzed by DNase, the methyl ASO or DNase when
moderately elevated titer: green is reduced and becomes sequelae of group A
240 Todd units adult colorless. streptococcal
320 Todd units child Tubes are graded for color. infection are
• ASO titers: • Normal titers: suspected, due to
increase 1-2 weeks after children 2-12 yrs 240-640 units occurrence of more
infection false positives and
peak 3-6 weeks ff the initial sx false negatives
 II. Avoiding antigen recognition by the host
MYCOPLASMA INFECTION -disguise, molecular mimicry (eg cross-reactivity between Ascaris Ag and human collagen)
CAUSATIVE AGENT CLINICAL MANIFESTATIONS - cover surface with host protein (eg Schistosomes; the adult worm takes up host red cell glycoproteins,
Mycoplasma pneumoniae belongs to the • The infection can be found in all age MHC molecules, and IgG)
- antigenic variation (eg Trypanosome & Plasmodium spp: switch to the expression of a new antigenic
class Mollicutes, which are unique groups, with an incubation period of 1-3
variant that these antibodies cannot recognize)
organisms because they lack cell walls and weeks. Symptoms include sore throat, III. Deviation of the host immune response
have a small genome, sterols in their cell chills, hoarseness, tracheobronchitis, and II. Avoiding antigen recognition by the host
-immunosuppression
membrane, and complex growth headache. -eg -disguise
trypanosomes: polyclonal activation of both T- and B-cell responses that diverts the immune response
requirements. • It can attach to cells in the respiratory away from specific
-molecular Ab production
mimicry
• It is the leading cause of upper tract and cause chronic inflammation. An -eg T. cruzi: secreted proline racemase (an enzyme that catalyzes the interconversion of l- and d-forms of
respiratory infections worldwide autoimmune response may play a role in proline)
III. Deviation of the host immune response
extrapulmonary complications. -manipulation of T-cell subsets: eg Filariasis: the cytokines that are induced by filarial infection, which
-immunosuppression
include TGFβ and IL-10, favor regulatory T-cell mediated responses
DETECTION OF ANTIBODIES TO Mycoplasma pneumoniae
• testing for cold agglutinins: presence in 50% of px with the infection (no longer used) IMMUNE
- helminths RESPONSES
polyclonally TO PARASITES
activate IgE-producing B-cells (good for the parasite, not good for the host) high
conc of irrelevant IgE binding to a mast cell will crowd out the parasite-specific IgE molecules and diminish
• Cold agglutinins: auto Ab -react with I/i Ag on RBC and cause their agglutination at temp the possibility of triggering the mast cell by specific antigen to initiate a protective defensive reaction
below 37˚C. Development of these Ab is thought to result from cross-reaction of the I humoral response develops when the organisms invade the bloodstream (malaria,
antigen on human RBCs or from alteration of the RBC by the organism. trypanosomiasis),
IMMUNE RESPONSES TOwhereas
PARASITES parasites that grow within the tissues (e.g., cutaneous
• SEROLOGICAL TESTS: EIA (most widely used), agglutination, IFA, complement fixation leishmaniasis)
humoral usually when
response develops elicit the
CMI, chronically
organisms invadeinfected host will (malaria,
the bloodstream be resistant to reinfection
trypanosomiasis),
whereas parasites that grow within the tissues (e.g., cutaneous leishmaniasis) usually
with fresh organisms, a situation termed concomitant immunity, seen in schistosomiasis elicit CMI,
• IgM, IgG, and IgA classes of Ab to M. pneumoniae may be detected.
chronically infected
and malaria host will be resistant to reinfection with fresh organisms, a situation termed
• EIA may be as sensitive as PCR. It is better to test for the presence of both the IgG and
concomitant immunity, seen in schistosomiasis and malaria
IgM classes of antibody for greater accuracy, but each class must be tested for separately. Humoral Immunity Cell-mediated Immunity
• IFA can also detect IgM or IgG classes. If the screening test is positive, then a serial ✓ Ab with right specificity present in adequate conc and affinity: ✓ Intracellular organisms, such as
dilution may be done to determine the concentration of antibody present. effective in providing protection against blood-borne parasites (eg. Toxoplasma gondii, Trypanosoma cruzi,
Trypanosoma brucei, sporozoite and merozoite stages of malaria) and Leishmania spp., use a variety of
✓ eg Trichinella spiralis: eosinophilia and high level of IgE Ab produced, ploys to subvert the macrophage killing
UNIT 2: Parasitic Diseases and their Serologic Tests serum levels of IgE can rise from normal values of 100 ng/mL to 10 000 systems. Cytokine-producing T-cells are
EVASIVE STRATEGIES BY PARASITES ng/mL (hallmarks of response to Th2type cytokines) crucially important for the stimulation
I. Resistance to effector mechanisms ✓ IL‐13 in the skin with IL‐4: switch factor for IgE production of macrophages to release their killing
(protection against schistosomes) power and dispose of the unwanted
T. cruzi DAF-like molecule accelerates decay of C3b ✓ Ag‐specific triggering of IgE‐sensitized mast cells leads to exudation of intruders.
Schistosoma SCIP1 molecule (schistosome C inhibitory protein 1) bind C9 inhibiting its serum proteins containing high conc of protective Ab in all the major Ig ✓ Organisms such as malarial plasmodia
mansoni polymerization & prevents formation of MAC classes and the release of eosinophil chemotactic factor. IgE can that live in cells that are not
facilitate ADCC toward schistosomula, the early immature form of the professional phagocytes may be
Plasmodium erythrocyte membrane protein 1 (PfEMP1) bind to CR1 (CD35): clustering eliminated through activation of
schistosome, and this can be mediated by eosinophils, monocytes,
falciparum of RBC= spread with minimal exposure to host immune system intracellular defense mechanisms.
macrophages, and platelets.
Trypanosoma surface Ag act as decoy proteins ✓ Schistosomula can also be killed by eosinophils using IgG for ADCC via ✓ induction of IFNγ and CD8+ T‐cells,
brucei binding through their FcγRII receptors to the IgG‐coated organism; the Interleukin‐12 and nitric oxide are also
Toxoplasma gondii inhibits phagosome–lysosome fusion by lining up host cell mitochondria major basic protein of the eosinophilic granules is released onto the required, and NK cells may play a
along the phagosome membrane parasite and brings about its destruction. subsidiary role by producing additional
✓ There may also be a localized requirement for Th1 cells given that IFNγ. Direct cytotoxicity by CD8+ T‐cells
Trypanosoma cruzi Escapes phagosome
IFNγ in the liver has been shown to be important in immunity to has been observed against hepatic cells
Leishmania Lipophosphoglycan protects them from oxidative burst by scavenging harboring malarial sporozoites.
schistosomes.
oxygen radicals. They also downregulate expression of MHC, CD80, and ✓ Eliminating worm infestations of the
✓ IgE‐mediated reactions may be vital for recovery from infection,
CD86, so diminishing T cell stimulation. gut requires the combined forces of
whereas the resistance in vaccinated hosts may be more dependent
- shedding and decoy systems are well suited to parasites or stages in the parasite life upon preformed IgG and IgA antibodies. cellular and humoral immunity to expel
cycle that are only briefly in contact with the immune system the unwanted guest. But the protective
strategy varies with the infection.
 MALARIA AMOEBIASIS SCHISTOSOMIASIS (bilharzia) TOXOPLASMOSIS
Causative 4 types of human malaria: • E. histolytica: presence of • S. mansoni, S. haematobium, and • Toxoplasma gondii: ubiquitous protozoan
agent: 1) P.falciparum (most common, chromatin on the nuclear S. japonicum parasite that infects humans by ingestion of
deadliest)) membrane • Adult male and female blood infective cysts (oocysts)
2) P. malariae flukes inhabit veins of the • Cats are known to be definitive hosts for this
3) P. ovale mesentery or bladder. parasite.
4) P.vivax (most common)
Mode of • bite of Anopheles mosquitoes Cysts: ingested • contact with free-swimming • Cysts hand-to-mouth contact from
Transmission: • seasonal, peak during and just trophozoites proliferate by binary infectious larvae that are released contaminated soil or cat litter or by ingesting
after the rainy season fission in the lumen of the colon. from an infected snail and bore into oocysts in raw or partially cooked pork, mutton,
Both cysts and trophozoites may be the skin, frequently while individuals or beef.
passed in feces, but only mature wade through contaminated water • blood transfusions or organ transplantation
cysts are infective. • Toxoplasma species can cross the placenta
Clinical • Malaria (from the Italian mal’ • Amebic dysentery: acute disease • Schistosomes survive in • disease is nearly always asymptomatic or may
Manifestations: aria, meaning “bad air”) is an acute characterized by bloody diarrhea unprotected blood vessels, because present with a mild lymphadenopathy
and sometimes chronic infection of with abdominal cramping. Invasion the invading form (schistosomula) • Invasion of CNS can be fatal, but occurs rarely,
the bloodstream characterized of the intestinal mucosa occurs, acquires RBC Ag that later help and typically occurs only in immunosuppressed px
clinically by fever, anemia, and producing ulceration that may lead protect the adult from attack by the • more common opportunistic infections seen in
splenomegaly. to perforation and peritonitis. immune system. individuals with AIDS.
• shaking chills, fever (up to 40° C • Amebic colitis: mimic ulcerative • Sx are initiated by the eggs: invade • fetus: exposed during the 1st trimester, death
or higher), generalized diaphoresis, colitis and other forms of tissues and cause hemorrhage nearly always occurs because of CNS damage
resolution of fever inflammatory bowel disease. Sx • chronic state can develop in which 2nd trimester: hydrocephaly, blindness, or other
paroxysm occurs over 6 to 10 hrs, generally are less severe than in the unexcreted eggs induce cell nervous system defects
initiated by the synchronous amebic dysentery but may include mediated DTH reactions, resulting in women exposed to this parasite before
rupture of erythrocytes with the non-bloody diarrhea, constipation, large granulomas that can obstruct pregnancy do not transmit the infection to the
release of new infectious blood abdominal cramping, weight loss the venous blood flow to the liver or fetus
stage forms known as merozoites • Amebic liver abscess: most bladder.
common form of extraintestinal
amebiasis, occurring in
approximately 5% of patients with a
history of intestinal amebiasis.
Sx: fever, right upper quadrant pain
Immunologic • immunity dev. over years of exposure & • Adult schistosome worms: T. gondii is capable of replicating inside human
Response never provides complete protection, decrease expression Ag on their macrophages. It can prevent the fusion of
reduce severe disease in malarial
outer membrane and enclosing lysosomes with phagosomes, hence can survive
infection
• no commercially available vaccine themselves in a glycolipid-and- indefinitely.
•vaccine candidates: glycoprotein coat derived from the
RTS,S/AS01 (for P. falciparum strain) host, masking the presence of their
FMP2.1/AS02:recombinant protein own Ag. Among the Ag observed on
based on apical membrane antigen 1 the adult worm are the host’s own
(AMA1) from 3D7 strain of P. falciparum
MSP3: merozoite surface protein 3
ABO blood-group and
histocompatibility Ag.
 MALARIA AMOEBIASIS SCHISTOSOMIASIS TOXOPLASMOSIS
Serologic • thick and thin blood films to • microscopic examination of fecal • Diagnosis: demonstrating eggs in feces •Diagnosis: examination of tissues, blood, or body fluids
demonstrate the intraerythrocytic specimens: easier to perform and or urine by direct wet mount or Demonstration of tachyzoites or tissue cysts is definitive.
Testing
parasites provide more definitive results formalin–ethyl acetate concentration
• Serology: primary approach in establishing a diagnosis of
compared to serum Ab testing methods. Eggs also may be detected in
toxoplasmosis in immunocompetent hosts.
• acridine orange staining and biopsies of rectal, bladder, and,
detection of parasite-specific DNA • Enzyme immunoassay (EIA) occasionally, liver tissues by crush • Sabin-Feldman dye test & IFA test: standards against which
provide enhanced sensitivity and identification of Entamoeba preparation or in histologic section. other methods are compared
specificity but generally are not employ enzyme conjugated to either
appropriate or available for smaller Ag or Ab, depending on the assay being • Serologic tests for negative urine or • Seroconversion to an antibody-positive state, a fourfold
laboratories performed. stool examination increase in the titer of IgG, or high levels of IgM and IgG Ab are
titers may persist for months or years considered diagnostic
• Immunocapture assays dev. for the after successful treatment • Although not widely available, a
• Enzyme immunoassays (EIA) for IgM, IgG, or IgA and indirect
detection of Plasmodium-specific Ag limited number of reference fluorescent Ab (IFA) assays for IgG: performed when
such as LDH or HRP-II appear to • PCR techniques laboratories and the CDC provide congenital toxoplasmosis is suspected.
provide a high degree of sensitivity and specificity is equal to microscopy testing. The CDC uses the Falcon assay • IFA testing- widely used
specificity for the diagnosis of more sensitive screening test in a kinetic enzyme-linked •EIA- method- sensitive, less difficult to perform, easier to
falciparum malaria easier to interpret, more difficult and immunosorbent assay (FAST-ELISA). interpret
time-consuming to perform
• Sensitive and specific IFA tests using • Sera that are positive by the screening • Elevated titers of IgG, IgM, or IgA Ab classes: infection has
occurred within the previous 3 to 9 months
Ag from the 4 human species are • Laboratories that do not use one of test are further evaluated by
available from the CDC. the immunologic or molecular methods immunoblot to improve specificity. • Elevated IgG titers w/o IgM Ab suggests an older infection.
to differentiate E. histolytica from E.
• A rapid EIA diagnostic test: allows dispar, and that rely exclusively on • Paired samples whose collection is separated by 3 weeks are
the differentiation of Plasmodium morphologic analysis, must use a tested to confirm the presence of recent infection. In a recent
falciparum from less virulent malaria reporting format that takes the infection, titers of both Ab classes will rise.
parasites differing technologies into
consideration. Thus, a report of “E. • Newborns with congenital toxoplasmosis usually have
detectable IgM antibody. Presence of elevated IgM titers in the
• Rapid testing: faster diagnosis and histolytica/E. dispar” would be most
mother’s serum further supports the diagnosis.
leads to earlier treatment appropriate in the latter circumstance.
• Unfortunately, these methods suffer • Currently, there are no useful serological procedures for
from inadequate sensitivity for diagnosing CNS infection in immunocompromised patients.
detection of lower parasite loads and
non-P. falciparum infections, and it is • PCR technology: highly sensitive and specific in detecting
therefore recommended that antigen- toxoplasmic encephalitis, disseminated disease, and
based assay testing be followed by intrauterine infection; testing is available from most reference
laboratories, the CDC, and select research laboratories. PCR is
confirmatory conventional thick and
now an important component of testing for pregnant women,
thin film examination. neonates, and immunocompromised hosts.
UNIT 3: Fungal Diseases and their Serological Tests IMMUNE RESPONSES TO FUNGI
A disease outcome is the result of the clash between the mechanisms of pathogenicity of the fungi
Fungus (pl. fungi) -eucaryotic organisms that are spore-bearing, have absorptive nutrition, and the mechanisms of resistance of the host, leading to the removal of the infection or its
progression according to the imbalance of these mechanisms.
lack chlorophyll, and reproduce sexually and asexually. Any fungal infection is called a
mycosis. Mycoses are classified into five groups according to the degree of tissue
Innate Immune Response Adaptive Immune Response
involvement and mode of entry into the host.
✓ skin, mucous membranes of respiratory, ✓ partially understood
gastrointestinal, and genitourinary tracts that provide ✓ cell-mediated immune
physical barriers that separate the host from the response plays the most
environment important role in the
✓ Pattern-recognition receptors (PRRs) (phagocytes, adaptive response to fungal
macrophages, and dendritic cells) initiate immune infections, role of humoral
response by sensing and recognizing the presence of immunity is much less
pathogen-associated molecular patterns (PAMPs) understood
present on the fungi. This event upregulates the ✓ cells of the innate
inflammatory response. immune system have been
✓ PRRs consist of 4 major classes: C-type lectin activated, cytokines and
receptors (CLRs), Toll-like receptors (TLRs), nucleotide- chemokines are produced
binding oligomerization domain (NOD)-like receptors, in response to the infection
and retinoic acid inducible gene I (RIG-I)-like receptors. Chemokines: recruitment of
✓ CLRs: for fungal recognition and induction of the T cells to the site of the
innate and adaptive immune responses to fungi. CLRs infection, aid in formation
important in fungal recognition include Dectin-1 and -2, of the adaptive immune
which recognize β-1,3 glucan and α-mannans, response (Th1 & Th2 cells)
FUNGAL SURVIVAL MECHANISMS
respectively, and mannose receptors, which recognize ✓ Once the T cells have
Fungi do not possess a large array of virulence factors that allow for them to be true
exposed mannose residues. Individuals with genetic committed themselves in
pathogens. Dev. of fungal infections occurs due to compromised immune responses.
deficiencies in CLRs are highly susceptible to fungal response to the fungi, they
Manifestations of fungal disease may range from unnoticed respiratory episodes to rapid,
infections. express an effector function
fatal dissemination of a violent hypersensitivity reaction.
Survival mechanisms: ✓ When PRRs bind fungi, they signal using their through the release of
intracellular tails or associated molecules (FcRγ) cytokines, primarily IFN-α,
• Presence of an antiphagocytic capsule
resulting in phagocytosis, initiation of killing TNF-α, and IL-17/22,
• Resistance to digestion within macrophages and destruction of phagocytes (e.g.,
mechanisms (e.g. production of reactive oxygen species) contributing to protective
neutrophils)
and also help drive the development of adaptive immunity to pathogenic
• Contain proteins that can interrupt the complement cascade on their surfaces (e.g., fungi.
immunity.
Candida albicans), thus mimicking the effects of the normal complement regulatory
✓Many CLRs use the same signaling molecule, CARD9
proteins C4bBP, CR1, and DAF (Caspase recruitment domain containing protein 9), to
• Ability to change morphotype by alteration of its surface glycans results in reduced host activate these antifungal immune responses. (eg mice,
recognition, reduction in inflammatory response, and increased virulence. or humans deficient in CARD9 are highly susceptible to
fungal infections, because although they have the PRRs
to bind fungi, the receptors can’t signal and therefore
there is no immunity) CARD9: most important molecule
for activating antifungal immune responses
Serological Test
Laboratory methods: culture “gold standard”. Others include the histopathological evidence of invasion, skin testing, and serological detection of Ag or Ab.

CRYPTOCOCCOSIS HISTOPLASMOSIS COCCIDIOIDOMYCOSIS ASPERGILLOSIS

(Valley fever, San Joaquin Valley fever,
and desert fever)
Description: • systemic mycosis caused by • facultative intracellular pathogen • Coccidioides immitis: previously • Invasive aspergillosis: most
Cryptococcus species complex Histoplasma capsulatum considered the sole etiologic agent of common life-threatening
coccidioidomycosis opportunistic invasive mycosis in
•Two species: • Subdivided into two varieties: immunosuppressed patients
-Cryptococcus neoformans: saprophyte ▪ H. capsulatum var. capsulatum • 2 genetically distinct Coccidioides clades:
with a worldwide distribution (aged, dried ▪ H. capsulatum var. duboisii: Limited to ▪ California clade (C. immitis) • Aspergillus fumigatus: most
pigeon droppings) equatorial Africa and causes a disease referred ▪ Non-California clade (C. posadasii) common group associated with
-Cryptococcus gattii (formerly called C. to as African histoplasmosis invasive aspergillosis, followed by the
neoformans var. gattii): tropical and ✓ Although a strong difference in Aspergillus flavus and Aspergillus
subtropical areas, eucalyptus trees, with • H. capsulatum in the soil is stimulated by bat genotype has been noted between these terreus
infection apparently limited in distribution, and bird guano, fungus sporulates and two species, the phenotypic differences
primarily to northern Australia and Papua produces conidia: generated aerosols, can be are minimal, thus making it difficult for • Invasive forms of Aspergillus most
New Guinea inhaled into the lungs most laboratories to distinguish between frequently begin in the lung, resulting
them. from inhalation of the conidia. The
• pathogens enter the host mainly through • Infections (> 90%) are self-limiting and organism grows and spreads in the
the lungs and has a predilection for asymptomatic. In a primary infection, the • Disease is contracted from inhalation of lung tissue. Invasive pulmonary
invading the CNS of the susceptible host organism disseminates to organs rich in soil or dust containing the arthrospores of Aspergillosis (IPA) may occur in the
mononuclear phagocytes. Coccidioides immitis. neutropenic px because of
•pulmonary infections (acute respiratory immunosuppression.
distress syndrome) are common • Organism can cause a potentially lethal • The disease may assume several forms,
infection in patients with preexisting primary pulmonary, primary cutaneous, •Disseminated aspergillosis may
•Cryptococcal meningoencephalitis: conditions. And causes opportunistic infection and disseminated. occur through hematogenous spread
primary life-threatening infection caused in patients with weakened immune systems. to distant sites or by contiguous
by C neoformans • Primary coccidioidomycosis is most extension from the lung.
• Clinical manifestations: frequently asymptomatic, as indicated by
• Cryptococcosis: approx 15% AIDS px -acute pulmonary infection the high prevalence of positive skin tests
-granulomatous and fibrosing mediastinitis for coccidioidal Ag in endemic areas.
• virulence and pathogenicity: capsule -chronic pulmonary histoplasmosis Disseminated infection most commonly
-consists of polysaccharide containing an -disseminated histoplasmosis through the affects the skin, the skeletal system, and
unbranched chain of alpha-1,3-linked reticuloendothelial system the meninges.
mannose units substituted with xylosyl and
β-glucuronyl groups
-effects of capsule: barrier to phagocytosis,
depleting complement, producing Ab
unresponsiveness, dysregulating cytokine
secretion, interfering with Ag presentation,
and enhancing HIV replication
 CRYPTOCOCCOSIS HISTOPLASMOSIS COCCIDIOIDOMYCOSIS ASPERGILLOSIS
Laboratory • Direct microscopic examination of the • Histological demonstration of yeast form • Direct microscopic methods (i.e., • Culture from sterile tissue with
Diagnosis: clinical material: India Ink in tissue Calcofluor white stain of respiratory histologic evidence of mycelial
• Fungal culture and biochemical tests • Fungal culture specimen and histopathology of tissue) invasion
• Matrix-assisted laser desorption • Fungal culture with specific molecular
ionization time-of-flight mass • Serological tests: probes for identification • Serological diagnosis is of limited
spectrometry (MALDITOFMS): rapidly ✓ detection of anti-Histoplasma Ab (i.e., utility because the
discriminate C. neoformans and C. gattii immunodiffusion and complement fixation) • Serological tests: immunosuppressed px will fail to
tests marginal for the diagnosis of mount an Ab response, even with
• Serological tests: histoplasmosis ▪ Hypersensitivity testing: intradermal invasive disease.
Ag tests take less time to perform and ✓ 2- to 6-week delay after exposure is injections as a screening test
are more specific than Ab detection required to produce Ab, reducing the value ▪ Immunodiffusion Antibody Test
of this testing for the diagnosis of acute ▪ Tube Precipitin Test (TPT) ✓ with reference antisera and
▪ Latex Agglutination (LA) Tests disease. Once patients are infected with H. ✓ One of the original methods for known Ag is a frequently used test
✓ detection of polysaccharide Ag of the capsulatum, Ab levels remain detectable for detecting C. immitis infection for the identification of Aspergillus
C. neoformans species complex in CSF many years. Ab to H. capsulatum and B. ✓ detection of IgM Ab against C. immitis spp. in almost all clinical types of
and serum dermatitidis also may cross react. Px with ✓ test involves overnight incubation of aspergillosis.
✓ Measurement of serum Ag appears to immune dysfunction may not produce the px serum with coccidioidal Ag
be a more sensitive test than testing of detectable levels of Ab and px with previous Formation of a precipitin button at the ✓ presence of 1 or more precipitin
CSF in HIV-infected patients. exposure might have elevated levels of Ab. bottom of a test tube demonstrates the bands suggests active infection
✓ Titration of positive results has been presence of IgM Ab precipitin bands correlate with CF
done traditionally to assess prognosis Complement Fixation (CF) Test A polysaccharide from the fungal cell titers
and to obtain a baseline for use in ✓ CF Ab, titer of 1:8 to yeast or mycelial Ag wall is used as the Ag in the test. In this test, the greater the number
following the effects of treatment. Large is considered positive and a titer of 1:32 is ✓ Tube precipitin Ab: detected in 90% of of bands, the higher is the titer. In
amounts of Ag and persistence of Ag indicative of an active infection px within the first 3 weeks of sx general, immunodiffusion measures
following therapy are poor prognostic ✓ Cross-reactions in the CF test occur in px ✓ Often referred to as the “IgM test” IgG and a positive result may
signs in px with aspergillosis, blastomycosis, or suggest past infection.
▪ Titers of 1:2 suggest infection, although coccidioidomycosis, but the titers are ▪ Complement Fixation (CF) Test
such findings have been found in px with ▪ Enzyme Immunoassay (EIA) Tests
usually lower. ✓ Most widely used quantitative
no evidence of cryptococcosis. serodiagnostic test for C. immitis ✓ detect IgE and IgG Ab
▪ Titers of 1:4 or higher are evidence of ▪ Immunodiffusion Technique infection ✓ detect Aspergillus galactomannan
an active infection. Higher titers also ✓ Precipitin band testing to determine the ✓ When mixed with a coccidioidal Ag, an Ag in serum
indicate more severe infections. presence of H and M Ab in the serum of immune complex is formed with the px
individuals. H and M Ab are produced Ab, resulting in depletion of ▪ ImmunoCAP
✓ False-positive: rheumatoid factor, against the H and M glycoproteins released complement. When the complement is ✓ newer method used to detect
other interference factors. Pretreating by mycelial and yeast phase cultures. depleted, Ab-coated RBCs added to the Aspergillus niger IgE in serum.
the specimen with heat and pronase, or ✓ H Ab are detected in < 10% of patients mixture fail to lyse. This test is often
2Mercaptoethanol, destroys these but, when present, signify active infection. referred to as the “IgG test” because IgG • Measurement of β-D-glucan (BDG)
factors and reduces the incidence of false ✓ M Ab are detected in up to 80% of is the immunoglobulin class usually in serum (a component of the cell
positive test results. individuals who have been exposed to the involved in the formation of this type of wall of most fungi)
fungus. These Ab may be found in immune complex.
individuals who have previously been
 ▪ Lateral Flow Immunochromatographic exposed to the organism or who have active ✓ IgM appears 1 to 3 weeks after
Assay (LFA) disease; thus, it is not useful in infection in 90% of symptomatic
✓ detection of cryptococcal Ag in fewer discriminating previous from current patients. IgG develops 3 to 6 months
than 15 minutes infection after the onset of symptoms.

serum CSF ✓ H and M bands appearing together o Titers of 1:2 to 1:4: presumptive
sensitivity: 100% Sensitivity: 99.3% indicate active infection. evidence of an early infection and
specificity: 99.8% Specificity: 99.1% should be repeated in 3 to 4 weeks.
✓ If only an M band is present, it indicates
early infection, chronic infection, or a o Titers of 1:8 to 1:16: evidence of active
▪ Indirect Immunofluorescence Assay recent reactive skin test. infection, particularly when
(IFA) accompanied by a positive
✓ Detects Ab to C. neoformans ✓ An H band appears later than the M band immunodiffusion test
✓ Most valuable when Ag tests are and disappears earlier.
negative and can even be combined with o Titers > 1:16 occur in 90% to 95% of px
an Ag test to determine a patient’s ✓ Disappearance of an H band suggests with disseminated coccidioidomycosis.
prognosis. regression of the infection.
▪ Enzyme Immunoassays (EIA)
✓ A positive test suggests a present or
recent infection ▪ Enzyme-Linked Immunosorbent Assay ✓ specific detection of IgM or IgG Ab
(ELISA)
✓ Performed in serum and urine for the ✓ positive EIA result: highly sensitive
detection of the polysaccharide antigen indicator for coccidioidal infection
✓ Urinary antigen tests have been shown to EIA tests for IgM and IgG are more
have a high sensitivity and specificity for sensitive than immunodiffusion tests,
detecting Histoplasma infections. however false positives have been
reported.

✓It is recommended that positive EIA

results, particularly positive IgM results,
should be confirmed with
immunodiffusion tube precipitin or
complement-fixing test results.

Serological testing for fungal diseases is less routine than serology for other infectious diseases because test availability is limited, and assays exhibit variable performance with respect to
sensitivity and specificity. Many of these tests are only offered by reference laboratories. With the rapid expansion of medical mycology, advances in molecular technology for prompt
identification of fungal pathogens are currently in play. Recent trends in molecular diagnostics of yeast infections are shifting from PCR to high-throughput technologies such as Next
Generation Sequencing (NGS) or Proteomics.