MEDICAL PARASITOLOGY  

     ○ Minimal overlap of cells and the erythrocytes maintain central

palor
●   50× oil immersion objective
      ○ To screen blood films for protozoa
01   TAXONOMY
●   100× oil immersion objective
Parasites are traditionally placed into one of two fundamental
      ○ For smallest parasites such as Plasmodium and Babesia
“kingdoms,” the Animalia (worms) and Protozoa. Parasites within each
species.
kingdom are then classified within hierarchical levels based primarily
●   Common mistake
on morphologic features and phenology.
      ○ Examine regions of the thin film where the blood is too thick or
  ● Phylum
too thin and the parasite morphology is distorted
  ● Class
Concentration Techniques
  ● Order
●   Buffy coat smears
  ● Family
      ○ Helpful in the detection of L. donovani, trypanosomes, and
microfilariae
02   LABORATORY METHODS
●   Knott’s concentration
Examination of Blood
      ○ For microfilariae detection when when the density of microfilariae
The most important techniques to be performed in the clinical
in peripheral blood is very low
laboratory to assist in the diagnosis of blood parasites
      ○ Anticoagulated blood is lysed with 2% formalin and centrifuged to
 ● Preparation
concentrate the microfilariae in the sediment
 ● Staining
      ○ Examined as a wet preparation or stained with Giemsa or
 ● Examination of thick and thin blood films
hematoxylin stain.
Parasites that may be detected in blood specimens include
●   Membrane filtration
 ● Malaria (Plasmodium spp.)
      ○ Blood is lysed and passed through a 5-μm membrane filter
 ● Babesiosis (Babesia spp.)
      ○ Stained with hematoxylin to reveal any microfilariae
 ● Trypanosomiasis (Trypanosoma spp.)
●   Fluorochrome acridine orange
 ● Leishmaniasis (Leishmania spp.)
      ○ Allows detection of blood parasite
 ● Filariasis (Wuchereria bancrofti, Brugia malayi, Loa loa, and
      ○ More sensitive than traditional thick and thin smears
Mansonella spp.).
      ○ Good in laboratories that encounter malaria frequently.
Thick and Thin Blood Films
Examination of Fecal Specimens
 ● Blood is spread over the slide in a thin layer, yielding intact, non-
 ●   Presence of intestinal parasites is identified by the use of:
overlapping cellular elements. Integrity of the blood cell membranes is
       ○ Direct examination of stool using wet mounts
important for determining the intracellular or extracellular nature of the
       ○ Concentration techniques
infection and the size of the infected erythrocyte.
       ○ Permanently stained smears
 ● in the thick film, blood is concentrated in a small area that is many
       ○ Culture (used less frequently)
cell layers deep
 ●   Intestinal protozoan infections
Preparation of slides
       ○ Diagnosed by detection of trophozoites, cysts, or oocysts.
●   Films should be prepared on clean, grease-free slides.
 ●   Newer immunoassay methods using species-specific antibody
●   Thick film
reagents
      ○ 1 to 2 small drops spread into an area of a dime (1.5cm)
       ○ Detects antigens of G. lamblia, Cryptosporidium spp., and E.
●   thin film
          histolytica
      ○ should be thin enough to be transparent.
 ●   Examination of three specimens collected every other day is
●   Dry in room temperature or a laminar blood flow hood to decrease
considered the minimum necessary to perform an adequate O&P
drying time.
evaluation.
●   Anticoagulants is discouraged when malaria is suspected due to
possible distortion of the parasites and interfere with staining.
Specimen Collection
●   Ethylenediaminetetraacetic acid (EDTA)
●   Specimens should not be collected for 1 week after the patient has
      ○ Transported to the laboratory within the hour to prevent
ingested any
deterioration of organism morphology
    materials that leave a crystalline residue, such as:
●   Blood is obtained by:
     ○ Nonabsorbable antidiarrheal compounds
      ○ Fingerstick
     ○ Antacids
            ■ Blood should flow freely to prevent dilution with tissue fluid
     ○ Bismuth
            ■ Should not be contaminated with the alcohol disinfectant
     ○ Barium
      ○ Earlobe puncture
     ○ Antimalarial agents.
      ○ Venipuncture
●   Oily laxatives such as mineral oil can also interfere with
            ■ First drop of blood (anticoagulant-free) from the needle is
examination
used, in bedside.
●   Antibiotics or contrast media may decrease the numbers of
organisms, especially protozoa, in the intestinal tract for several weeks
Staining
●   Collected directly into clean, dry containers, or onto a specially
●   Giemsa stain
designed wax or plastic collection sheet
     ○ Allows visualization of erythrocyte inclusions that occurs with
     ○ Urine or toilet water in the specimen may destroy protozoal
infection by certain malarial parasites.
trophozoites
     ○ Requires more attention to preparation of reagents and staining
     ○ Water or soil can introduce free-living organisms
protocol
●   Diarrheic specimens
     ○ Must be made each day by diluting stock solution into phosphate
     ○ Clean bedpans
buffered water (pH 7.0 to 7.2)
Specimen Handling
●   Wright’s stain
●   Containers
     ○ Can be used for thin films
     ○ Tight-fitting lids
     ○ Thick films must be lysed in water before staining because
     ○ Placed in plastic bags before transport to the laboratory
Wright’s stain incorporates alcohol as its fixative
●   Specimens are submitted to the laboratory fresh or in appropriate
     ○ Can be automated
preservatives
●   All fresh specimens
Examination of Smears
     ○ Should be examined within 1 hour of passage
●   Thick and thin smears are viewed at under the low power (10×)
     ○ Liquid specimens should be examined within 30 minutes or
objective to detect microfilariae.
placed immediately in preservatives to maintain the best yield.
●   Feathered edge of thin smears
Specimen Preservation
      ○ Microfilariae are often carried there during preparation.
●   Specimens that cannot be processed immediately should be left at
room temperature or refrigerated
●   Specimens should not be placed in an incubator as it only speeds       ○ Lighter parasite cysts and eggs rise to the surface of a solution of
disintegration of parasites. high specific gravity
      ○ Zinc sulfate centrifugal flotation technique of Faust, most widely
used concentration procedures in the United States
●   Two-vial technique
     ○ One portion of specimen is fixed in three parts of 5% to 10% Concentration Techniques: Formalin ether sedimentation
buffered formalin and another portion in three parts of polyvinyl alcohol ●   A biphasic sedimentation technique that is efficient in recovering
(PVA) fixative most protozoan
●   Sodium acetate–formalin (SAF)     cysts and helminth eggs and larvae, including operculate eggs
     ○ Can be used for permanent stains as well as for direct mounts ●   Moderately effective for schistosome eggs
and concentration procedures ●   Cons
     ○ Does not contain mercury, which is present in Schaudinn’s and      ○ Hymenolepis nana may be missed
PVA fixatives      ○ Concentrations of G. lamblia and Iodamoeba bütschlii cysts may
●   Merthiolate-iodine-formalin (MIF) not be very good
●   Attention must be paid to the recommended speed and time of
Macroscopic Examination centrifugation
●   Examined grossly for:
     ○ Consistency (formed, soft, loose, or watery). Concentration Techniques: Zinc sulfate centrifugal flotation
     ○ Presence of mucus, blood, larval or adult worms, and proglottids.  ●   Fresh stool is processed using zinc sulfate with a specific gravity of
●   Watery or loose specimens 1.18
     ○ Protozoan trophozoites predominate  ●   Formalinized stool is processed with a solution of specific gravity of
●   Formed or soft specimens 1.20
     ○ Cysts predominate  ●   Yields a cleaner preparation than is provided by formalin-ethyl
acetate concentration
Microscopic Examination  ●   Unreliable for the recovery of:
●   Direct saline wet mounts of fresh feces        ○ Nematode
     ○ Allow detection and observation of motile protozoan trophozoites        ○ Larvae
and helminth larvae        ○ Infertile eggs of Ascaris
●   Direct mounts of preserved feces        ○ Eggs of most trematodes and large tapeworms
     ○ Allow detection of parasites that do not concentrate well.  ●   Recovery problems stems from stool specimens containing
●   Concentration procedures excessive amounts of fats
     ○ Increase the examiner’s ability to detect protozoan cysts and  ●   Use of formalinized stool specimens rather than fresh stool helps
helminth eggs and larvae clear the specimen and prevents popping of opercula and distortion of
     ○ Unsatisfactory for detecting protozoan trophozoites the parasites
●   Permanent stains
     ○ For detection and morphologic examination of protozoan Permanent Stain
trophozoites and cysts ●   Pros:
●   Fresh soft, loose, or watery specimen      ○ Provides a permanent record of a patient’s specimen and allows
     ○ Should have all three procedures performed review
●   Watery specimens      ○ Is the only one designed for analysis using the oil immersion
     ○ Concentrated by simple centrifugation rather than by flotation or objective (100×)
formalin–ethyl acetate concentration      ○ Most useful for detection of protozoal trophozoites and cysts
●   Specimens in preservatives      ○ Inherently more sensitive for detecting protozoal infections
     ○ Direct wet mount may be omitted ●   Cons
●   Formed specimens      ○ Not useful for detecting helminth eggs or larvae
     ○ Examined by a concentration procedure ●   Recommended for every stool sample submitted for O&P
     ○ Improved yield when a permanent stain is added to the workup examination
●   Wheatley’s Trichrome Stain
Direct Wet Mount      ○ All-purpose method that allow detection of amebae and flagellates
●   Most easily performed parasitologic test      ○ Specimens::
●   Most useful when fresh specimens, especially liquid stools or          ■ Schaudinn’s fixative or PVA fixative; SAF- or MIF-preserved
duodenal aspirates, are   examined for motile trophozoites or helminth          ■ Preserved using single-vial commercial fixatives may also be
larvae stained with this or a slightly modified protocol
●   A small amount of stool is mixed with a drop of 0.85% saline and ●   Iron Hematoxylin Stain
covered with a coverslip.       ○ All-purpose method that allow detection of amebae and
●   Should be dense enough that newspaper print can just be read flagellates
through it.       ○ Enhanced definition of key nuclear and cytoplasmic
●   Examination of the entire coverslip is performed systematically characteristics
under the low-power       ○ Incorporates carbol fuchsin, allowing concurrent staining of acid-
    (10×) objective fast organisms like:
●   Suspicious objects and those that are refractile, such as protozoan            ■ Cryptosporidium
cysts, should then be examined with the high-power (40×) objective            ■ Cyclospora
●   Detection of motility of slow-moving amebae is required to be            ■ Cystoisospora
examined for at least 15 seconds ●   Modified Acid-Fast Stains
Concentration Techniques       ○ Can detect oocysts of Cryptosporidium, Cyclospora, and
●   Performed on fresh or preserved specimens Cystoisospora which are
●   More sensitive than direct wet mount examination for detection of          difficult to recognized in the two previous stains.
protozoan cysts and helminth eggs and larvae because:       ○ Sensitive and cost-effective for detection of these protozoa
      ○ Decreases the amount of background material       ○ Lack specificity
      ○ Concentrates the organisms       ○ The use of positive control material is mandatory
●   Based on sedimentation or flotation principles ●   Stains for Microsporidia
●   Sedimentation      ○ A modified trichrome stain using an increased (10-fold)
      ○ Heavier parasites settle to the bottom as a result of gravity or concentration of chromotrope 2R combined with an increase in staining
centrifugation time has gained acceptance as a specific test for the identification of
      ○ Formalin ether sedimentation method of Ritchie, most widely microsporidial spores.
used concentration procedures in the United States      ○ Uvitek-2B and Calcofluor white
●   Flotation            ■ For rapid and sensitive screening of stool and other clinical
specimens for such spores
           ■ Fungi will also be highlighted        ○ When amebae are suspected, permanent stains should
           ■ The small size (1.5 to 3 μm) of these organisms makes their be performed
detection difficult, and such studies should not be undertaken without        ○ Acid-fast or specific antibody-based stains to detect
appropriate control materials for comparison

Additional Techniques for Examination of Enteric Parasites Cryptosporidium oocysts

Cellulose Tape Technique for Pinworms        ○ Trichrome or fluorochrome stains for detection of spores of
●   Enterobius vermicularis, migrates from the cecum to the perianal microsporidia
skin, where the female deposits typical eggs that are fully   ●   Aspirates
embryonated.        ○ Giemsa staining is often appropriate when examining for
●   Eggs or, occasionally, adult worms may be detected on pressed protozoa, especially the hemoflagellates
adhesive cellophane tape on to the perianal skin.   ●   Biopsies
●   Collected in the morning before bathing and defecation.        ○ Examined by compressing the fresh specimen between two
glass slides
Egg Studies        ○ Muscle biopsy specimens for Trichinella species larvae
●   Estimation of worm burden occasionally is requested to assist in the        ○ Rectal or bladder biopsies may be examined for schistosome
evaluation of therapeutic efficacy eggs
●   Procedures include:
      ○ Direct smear method of Beaver PARASITE CULTURE TECHNIQUES
      ○ Stoll dilution egg count  ●   For a wide variety of protozoan parasites
      ○ Kato’s thick smear  ●   Infrequently requests
●   Levels of egg counts indicating clinical significance vary, depending  ●   Usually for:
on the infecting species and the person’s age and nutritional status        ○ T. vaginalis
●   Egg-hatching methods for schistosomiasis        ○ Leishmania spp.
      ○ Urine or stool is mixed in about 10 volumes of water, which is        ○ Trypanosoma cruzi
then placed in a sidearm or Erlenmeyer flask. All but the sidearm or the        ○ E. histolytica
top of the flask is covered with foil wrap, and the unit is placed under a        ○ Acanthamoeba spp.
desk lamp        ○ Naegleria fowleri
      ○ Hatched miracidia are positively phototropic and congregate near
the light. Eggs, if available, may be examined directly for viability by IMMUNODIAGNOSTIC METHODS
examining for movement of cilia within flame (excretory) cell  ●   Identifies the parasitic antigen or the antibody that is produced in
Nematode Culture and Recovery Techniques response to the parasitic infection
●   In all culture methods, feces are incubated in a humid environment        ○ Enzyme immunoassay (EIA)
to encourage egg hatching        ○ Indirect immunofluorescence assay (IFA)
●   Harada-Mori and filter paper/slant techniques        ○ Direct fluorescence antibody assay (DFA)
      ○ Larvae migrate from the feces into a water phase, where they        ○ Western blot
may be readily detected        ○ Radioimmunoassay
●   Charcoal culture        ○ Immunodiffusion
      ○ Larvae first migrate into a dampened gauze pad, which is then Antigen Detection
placed in water, allowing the larvae to settle out. ●   Useful for initial testing or in instances in which traditional tests are
●   The Baermann funnel technique and agar culture negative, yet a high index of clinical suspicion remains
      ○ Sensitive and reliable methods for recovery of Strongyloides and ●   Stool samples
other nematode larvae from a stool specimen       ○ Usually performed using fecal immunoassays
      ○ Examinations over 1 week’s time using a concentration technique            ■ Easy to use and rapid, permit batch processing, and do not
may be required to detect the infection of latent Strongyloides require experienced microscopists
Nematode Culture and Recovery Techniques       ○ Marketed for:
●   The Baermann funnel technique            ■ G. lamblia
     ○ Feces are placed on several layers of gauze on top of a wire            ■ C. parvum/C. Hominis
screen that is suspended in a funnel.            ■ E. histolytica/E. dispar group, and E. histolytica
     ○ The bottom of the funnel is clamped off, and water is added to the ●   Blood or serum
level of the gauze.       ○ Available for Plasmodium spp. and W. bancrofti
     ○ Larvae actively migrate through the gauze and settle to the ●   T. vaginalis antigen detection
bottom of the funnel, where they may be drawn off for examination       ○ Latex agglutination test on viginal swabs
     ○ Used infrequently in the clinical laboratory due to being labor       ○ Rapid antigen tests on vaginal samples sensitivities comparable
intensive with those of culture but still less than nucleic acid amplification tests
●   The agar culture technique Antigen Detection
     ○ Feces are plated on a nutrient agar and incubated at room ●   Rapid antigen detection methods
temperature for several days.      ○ Malaria
     ○ Over time, the larvae will migrate out of the feces into the agar           ■ may detect histidine-rich protein II (HRP-II), parasite lactate
and carry fecal bacteria with them. dehydrogenase (pLDH), parasite aldolase, or a combination of these
     ○ Growth of the bacteria in the larval tracks facilitates identification antigens in peripheral blood
of larvae in the specimen.      ○ HRP-II tests specifically for Plasmodium falciparum
     ○ pLDH and aldolase tests detect all four Plasmodium spp.
Objects Resembling Enteric Parasites ●   Enzyme immunoassay (EIA)
●   A large variety of objects that closely resemble various parasite life      ○ Available in microwell format
cycle stages      ○ Accepted samples are frozen, fresh, or 10% formalin–preserved
      ○ White blood cells, macrophages, and squamous and columnar stool samples
epithelial cells may resemble amebae      ○ Parasite antigen is captured by immobilized antibodies coated on
      ○ Yeasts and starch granules may resemble protozoal cysts microwells and is detected by an enzyme-conjugated secondary
      ○ Pollen and fungal conidia may resemble helminth eggs antibody that is capable of producing a colored reaction following the
      ○ Plant fibers may resemble nematode larvae addition of substrate.
      ○ Pieces of vegetables or vegetable skins may resemble adult Antigen Detection
worms or proglottids ●   Lateral flow cartridges
     ○ A popular format of immunoassay because of their ease of use
EXAMINATION OF UROGENITAL AND OTHER and the minimal performance time required
SPECIMENS (SPUTA, ASPIRATES, BIOPSIES)      ○ Stored conveniently at room temperature used for single or batch
  ●   Sputa processing
       ○ Wet mount
     ○ The parasite antigen in the sample migrates through the
membrane and binds to specific capture antibodies; use of a
secondary reagent results in development of a colored reaction
     ○ Only the supernatant of a well-mixed stool sample is used to
ensure complete migration of the specimen
     ○ Any color visible at the reagent test zone (usually a band) is
interpreted as positive
Antigen Detection
●   Direct Fluorescent Antibody testing (DFA)
     ○ Allows direct visualization of the parasites in stool specimen using
antibodies conjugated to fluorescent dyes.
     ○ Kits are available for detection of cysts of G. lamblia and oocysts
of Cryptosporidium spp
     ○ Fresh stool samples can be tested directly, the sensitivity of the
assay can be improved by performing the test on centrifuged stool (500
g for 10 minutes).
Serologic Diagnosis
●   Used as an adjunct to the usual diagnostic modalities or in special
situations wherein identification of the parasite or its antigen or nucleic
acid from host tissue or excreta is not possible
      ○ Toxoplasmosis and toxocariasis reside in deep tissue
      ○ Cysticercosis and echinococcosis develop in organs invasive
studies that may be required are not recommended in the initial patient
evaluation
      ○ Diagnoses:
           ■ Extraintestinal amebiasis (e.g., amebic liver abscess) and
trichinellosis
           ■ Chronic stages of trypanosomiasis
           ■ Occult infections such as visceral larva migrans, cysticercosis,
and filariasis
      ○ Understanding of the epidemiology of diseases:
           ■ Schistosomiasis, toxoplasmosis, amebiasis, and babesiosis
           ■ Chagas disease and malaria
●   Tests for parasitic diseases generally evaluate IgG levels, with the
exception of toxoplasmosis and babesiosis
      ○ IgM- and IgA-specific antibodies may be helpful for determining
the age of infection.
      ○ IgM and IgA may persist for as long as 2 years after the primary
infection,which complicates interpretation of positive test results for
these immunoglobulins.
      ○ Detection of antibodies, especially IgG, provides evidence of
infection but may not be able to differentiate active from past exposure
●   Developed in-house
      ○ Lack correlation with universal standards
      ○ Interpretive criteria are established by reagent manufacturers
      ○ These makes the criteria often vary from institution to institution
      ○ Should know performance characteristics, including sensitivity
and specificity,
      ○ Should be aware that cross-reactions may occur
           ■ Antibody tests for Chagas disease cross-reacts with
Leishmania infections
           ■ Helminthic parasites are well known to cross-react in
serologic assays that use crude antigen preparations because of
phylogenetic, hence antigenic, similarities.
Serologic Diagnosis
●   Several factors that may influence the test performance
     ○ Disease manifestation, test format, reagents used, and parasite
viability
     ○ Parasite viability; hydatid cysts occurring in the lung and dead or
calcified cysts are less frequently detected than active cysts in the liver
●   Sensitivity
     ○ Increased in patients with invasive amebiasis
     ○ Weak in intestinal amebiasis with minimal tissue invasion
     ○ Absent for asymptomatic carriers
MOLECULAR DIAGNOSTIC METHODS
 ●   Diagnostic methods using DNA and RNA amplification and nucleic
acid probe techniques
      ○ Offer high levels of sensitivity and specificity
 ●   Advantages
      ○ Rapid (automated) results, high sensitivity and specificity
      ○ The ability to detect and differentiate species variants all
independent of the patient’s underlying immune status
           ■ A potentially limiting feature of serologic assays
      ○ High availability due to the introduction of multiple commercial
assays
           ■ Some of which are FDA cleared for in vitro diagnostic use
           ■ Tests for T. vaginalis, G. lamblia, Cryptosporidium spp., E.
histolytica, and Cyclospora cayetanensis
      ○ May be used to monitor the success of antiparasitic therapy or to
detect reactivation following therapy
 ●   Disadvantages
           ■ Prone to cross-contamination if proper processing precautions
are not strictly enforced
MOLECULAR DIAGNOSTIC METHODS
 ●   Nucleic Acid Amplification Assays (NAAT)
       ○ Some are available in real-time format
            ■ Kinetics of the nucleic acid amplification reaction is recorded
and analyzed by computer algorithms to allow detection of amplicons
                 ● Allows for rapid detection
                 ● Lessens the risk for amplicon cross-contamination
       ○ For parasites such as Plasmodium spp., Babesia spp.,
Leishmania spp.,
          T. gondii, and Trypanosoma spp.
 ●   Isothermal methods
       ○ Strand displacement amplification, transcription-mediated
amplification, and loop-mediated isothermal amplification (LAMP)
 ●   Nucleic Acid Amplification Assays (NAAT)
QUALITY ASSURANCE, QUALITY IMPROVEMENT, AND SAFETY
  ●   Quality assurance
       ○ A well-written and complete procedure manual that is reviewed
annually
       ○ Guidelines for maintaining all specimen and test result records
       ○ A complete QC program with appropriate technical supervision
and review
       ○ Participation in an approved proficiency testing program
       ○ Competency assessments should be up to date
       ○ Reference materials should be readily available
  ●   Quality improvement
       ○ Focus on customer satisfaction, using a variety of available
measures, and to participate in the team approach to identifying
problems and generating solutions as part of a continuous quality
improvement process
  ●   Safety
       ○ Unpreserved specimens for parasitologic examination should be
considered potentially infectious
       ○ All blood and body fluids should be handled according to
Standard Precautions as defined by the Final Rule on Blood-borne
Pathogens by the Occupational Safety and Health Administration
       ○ Strict observance of proper specimen handling techniques and
disposal is essential.
       ○ Personal attention to hand washing is also necessary
       ○ Use of ethyl acetate in place of ether in the performance of
concentration techniques is strongly recommended to guard against
the possibility of explosion
03 BLOOD AND TISSUE PROTOZOA
PLASMODIUM SPECIES (MALARIA)
 ●   Malaria (from the Italian mal’ aria, meaning “bad air”) is an acute
and sometimes chronic infection of the bloodstream
 ●   Characterized clinically by:
       ○ Fever, anemia, and splenomegaly, and is caused by
apicomplexan parasites of the genus Plasmodium.
 ●   Clinical features
       ○ Shaking chills, fever (up to 40° C or higher), and generalized
diaphoresis, followed by resolution of fever.
 ●  The paroxysm occurs over 6 to 10 hours and is initiated by the
synchronous rupture of erythrocytes with the release of new infectious
blood stage forms known as merozoites
 ●   Spread exclusively by female anopheline mosquitoes
 ●  The four main species of plasmodium causing human malaria are
     Plasmodium vivax, P. falciparum, Plasmodium malariae, and
Plasmodium ovale
 ●   Plasmodium vivax (17%)
      ○ Tropical and temperate zones
      ○ People who lack certain Duffy blood group determinants are
protected to some extent against P. vivax infection
 ●   P. falciparum (58%)
      ○ Tropical areas worldwide
      ○ People with sickle cell trait are less susceptible to P. falciparum
malaria
 ●   Plasmodium malariae (3%)
      ○ Occurs worldwide but lesser than Plasmodium vivax & P.
falciparum
 ●   Plasmodium ovale (3%)
      ○ Least frequent of the malarias, with most cases being acquired in
western Africa, India, or South America
●   Undetermined (17%), with 1% having infection with two species
●   Common presenting symptoms
     ○ Chills and fever, which often are associated with splenomegaly
     ○ Febrile episodes occur irregularly but eventually become more
synchronous
     ○ Anemia, diarrhea, abdominal pain, headache, and muscle aches
and pains
     ○ Peripheral smears may show leukocytes that contain malaria
pigment (hemozoin).
     ○ Increased reticulocyte counts occur commonly and are associated
with rapid erythrocyte turnover
          ■ Greatly enlarged platelets may occur on peripheral blood films
●   Plasmodium ovale & Plasmodium vivax
     ○ Infection become symptomatic within 6 months of exposure
     ○ May have relapses after many months or, occasionally, years
     ○ Require treatment with primaquine to eradicate hepatic
hypnozoites and to prevent relapse
Clinical Disease
●   P. falciparum
     ○ Infection become symptomatic within 1 month of exposure
     ○ High rates (50%) of parasitemia which leads to severe hemolysis
with hemoglobinuria and profound anemia
     ○ Occlusion of vessels due to schizonts sequestered in small
vessels of the body
            ■ Causing symptoms related to capillary obstruction and tissue
anoxia
     ○ Brain involvement is known as cerebral malaria, in which the
patient becomes disoriented progressing to delirium, coma, and often
death.
     ○ Most fatal case of malaria
     ○ May have symptom free periods but suffer from sporadic
recrudescences owing to persistent low-grade parasitemia
     ○ Widespread appearance of resistance against chloroquine and
other antimalarials
●   Plasmodium malariae
     ○ Infection become symptomatic within 6 months of exposure
     ○ ay have symptom free periods but suffer from sporadic
recrudescences owing to persistent low-grade parasitemia
Diagnosis
●   Is always be included in the differential diagnosis of fever in patients
who have a history of travel to or residence in endemic areas
●   Parasites in thick and thin blood films
      ○ Preferably thick films due to a greater amount of blood is being
examined
●   Blood specimens are collected just before the next anticipated fever
spike or at the onset of fever.
●   Schüffner’s stippling still may be a helpful identifying characteristic,
and it may be recognized around growing trophozoites as a pink halo
rather than as distinct granules seen in thin films
●   Three major factors should be considered:
      ○ Appearance of infected erythrocytes
      ○ Appearance of parasites
      ○ Stages found
Serologic Diagnosis
BABESIA SPECIES (BABESIOSIS)
 ●   Etiologic agent: apicomplexan protozoa
 ●   Vector: “black-legged” or “deer tick”
 ●   Human infection is predominantly in the northeastern and
midwestern states, where the rodent parasite Babesia microti is
responsible for infection
 ●   Rarely transmitted by blood transfusion and transplacentally
 ●   The spectrum of babesiosis varies from latent, subclinical infection
to fulminant, hemolytic disease
 ●   Fatalities are mostly splenectomized or immunocompromised
individuals
 ●   Immunocompetent persons experience symptoms similar to
malaria
      ○ Fever, chills, malaise, and anemia, although without recognizable
periodicity
 ●   Babesia parasites multiply in erythrocytes by binary fission
producing morphologically indistinguishable trophozoites and
gametocytes
      ○ Trophozoites appear pear-shaped
      ○ Presence of multiple rings in one cell forming a tetrad (Maltese
cross)
      ○ Have a heterogeneous appearance with round, oval, spindled,
and “racket”
      ○ Babesia-infected cells lack hemozoin pigment, which is present
in Plasmodium-infected cells
Hemoflagellates Trypanosoma
●   Trypanosoma brucei (African or Old World trypanosomiasis)
      ○ 30 μm long with graceful curves and a small kinetoplast
      ○ Vector: Tsetse flies Glossina
      ○ East African trypanosomiasis caused by T. brucei rhodesiense
           ■ Rapidly progressive acute febrile illness with
lymphadenopathy
           ■ Patients die before central nervous system involvement is
prominent
      ○ Western African trypanosomiasis is caused by T. brucei
gambiense
           ■ African sleeping sickness
           ■ Chronic, begins with intermittent fevers, night sweat and
malaise.
           ■ Lymphadenopathy in the posterior cervical lymph nodes
Trypanosoma
●   T. cruzi (American or New World trypanosomiasis, or Chagas
disease).
      ○ 20 μm long and display a larger kinetoplast
      ○ Trypomastigotes commonly assume a C shape
      ○ Vector: kissing bugs of the family Reduviidae
      ○ Occurs in the United States, Mexico, Central America, and most
of South America
      ○ Acute disease
            ■ Most common in children younger than 5 years of age
            ■ Characterized by malaise, chills, fever, hepatosplenomegaly,
and myocarditis.
      ○ Chagoma
            ■ Swelling of tissues following the bite of an infected reduviid
●   Trypanosoma rangeli a third specie that does not cause clinical
illness
●   Diagnosis
      ○ High total IgM levels in blood and cerebrospinal fluid
      ○ Demonstrating the parasites on:
          ■ Thick and thin films of peripheral blood, buffy coat
preparations, or aspirates
              of lymph nodes or bone marrow, or in spun CSF that is
stained with Giemsa
      ○ Culture or animal inoculation may be helpful if it is available
Leishmania
●   Disease of the reticuloendothelial system caused by kinetoplastid
protozoa of the genus
    Leishmania
●   Vector: Sandflies genera Phlebotomus (Old world) and Lutzomyia
(New World)
●   Cutaneous Leishmaniasis
     ○ Old world - “Oriental sore” occurs in southern Europe, northern
and eastern Africa, the Middle East, Iran, Afghanistan, India, and
southern Russia
           ■ Leishmania tropica - produces the urban or dry ulcer, may be
viscerotropic
           ■ Leishmania major - rural or wet ulcer
           ■ Leishmania aethiopica - mucosal lesions or diffuse cutaneous
leishmaniasis
                ● Characterized by multiple skin nodules resembling
lepromatous leprosy
           ■ Also L. donovani and Leishmania infantum
     ○ New world
           ■ Leishmania mexicana - involves the earlobe (chiclero ulcer),
self limiting
           ■ Leishmania amazonensis - produce diffuse cutaneous lesions
similar to those produced by L. aethiopica
           ■ Leishmania peruviana - an infection called uta, benign
cutaneous lesion that occurs predominantly in children, from domestic
dogs
●   Mucocutaneous Leishmaniasis
     ○ Caused primarily by L. braziliensis and species in the Viannia
subgenus
     ○ Produce typical cutaneous lesions that generally are more
aggressive, last longer, and often disseminate to mucous membranes,
especially in the nasal, oral, or pharyngeal areas
           ■ may produce disfiguring lesions secondary to erosion of soft
tissues and cartilage
     ○ Mexico and Central and South America
●   Visceral Leishmaniasis
     ○ Old world
          ■ Caused by L. donovani or by L. infantum. L. donovani, L.
infantum
     ○   New world
           ■ Caused by L. chagasi
     ○   Infection is called kala-azar
     ○   Usually benign and often subclinical
●   Diagnosis
     ○ Established by visualization of amastigotes in smears, imprints, or
biopsies, or by growth of promastigotes in culture
         ■ Their size (2 to 4 μm) smaller in tissue sections due to
shrinkage during fixation
     ○ A culture is desirable because it is more sensitive and allows
determination of the species or subspecies
Toxoplasma Gondii
●   T. gondii is a protozoan parasite of the phylum Apicomplexa
●   Definitive hosts: felines
      ○ It’s life cycle is completed in the intestinal epithelium
●   Acquisition: by ingestion of inadequately cooked meat, especially
lamb or pork, that contains tissue cysts or by ingestion of infective
oocysts from material contaminated by cat feces
      ○ Transmission by inhalation, blood transfusion, organ
transplantation, and transplacentally to the developing fetus also can
occur
●   Acute infections
      ○ Are asymptomatic or mimic other infectious diseases in which
fever and lymphadenopathy are prominent
●   Immunocompromised individuals
      ○ May manifest CNS involvement
●   Other manifestations:
      ○ Pneumonitis, myocarditis, retinitis, pancreatitis, or orchitis
●   Diagnosis
     ○ Difficult to diagnose clinically and is often discovered at autopsy
     ○ By examination of tissues, blood, or body fluids
         ■ Tachyzoites or tissue cysts is definitive
              ● Fluorescence or immunohistochemical stains are useful
              ● Giemsa is good for staining smears
     ○ PCR technology is highly sensitive and specific in detecting
toxoplasmic encephalitis, disseminated disease, and intrauterine
infection
     ○ Serology is primary approach for immunocompetent hosts
OPPORTUNISTIC FREE -LIVING AMEBAE
Naegleria fowleri
●   Typically affects children and young adults who have been
swimming, jumping, or diving in warm freshwater lakes or pools
●   Amoeboflagellate enters the brain via the cribriform plate and
olfactory bulbs
     ○ It produces an acute hemorrhagic meningoencephalitis that is
usually fatal within 1 week of onset of symptoms upon reaching frontal
lobes.
●   Extremely poor prognosis
●   Diagnosis:
     ○ Autopsy examination by the finding of trophozoites (cysts are
rarely seen) in tissue sections.
     ○ Identifying typical trophozoites in CSF on direct wet mounts, in
stained preparations, or in culture
           ■ Trophozoites measure 10 to 35 μm; have large round, central
karyosomes and if exposed to warm distilled water, convert to
flagellated forms in 1 to 2 hours
           ■ Cysts are spherical, measuring 7 to 15 μm in diameter
Acanthamoeba
●   Granulomatous amebic meningoencephalitis (GAE)
      ○ Caused by Acanthamoeba castellani, Acanthamoeba culbertsoni,
          Acanthamoeba polyphaga, and Acanthamoeba astronyxis
●   Usually a subacute or chronic opportunistic infection of chronically
ill, debilitated, and immunosuppressed individuals
      ○ Leads to death weeks to months following onset of symptoms
      ○ Individuals with AIDS and may present as ulcerative skin lesions,
subcutaneous abscesses, or erythematous nodules
      ○ Spreads hematogenously from primary foci in skin, pharynx, or
the respiratory tract
●   Readily become airborne and may be recovered from the throat and
nasal passages
●   The pathologic reaction in tissues is granulomatous, with
trophozoites predominating in viable tissue, and cysts predominating in
areas of necrosis
●   Diagnosis
      ○ Established at autopsy
      ○ Brain biopsies or recovered using the culture technique
described for Naegleria
            ■ 15 to 45 μm, and display needlelike filamentous projections
from the cell
Balamuthia & Sappinia
●   Granulomatous amebic meningoencephalitis (GAE)
     ○ Caused by Balamuthia mandrillaris
●   Diagnosis:
     ○ Identified using specific monoclonal or polyclonal antisera in DFA
or immunoperoxidase assays
●   Encephalitis can be caused by Sappinia pedata
04 INTESTINAL AND UROGENITAL PROTOZOA AMEBAE AND
BLASTOCYSTIS HOMINIS
Entamoeba histolytica
●   Acquired by ingestion of contaminated food or water
●   Diagnosis:
     ○ Examination of a series of stool specimens should be sufficient for
diagnosis of intestinal amebiasis in most cases
     ○ Cysts of E. histolytica are spherical and measure 10 to 20 μm
     ○ Trophozoites vary from 10 to 60 μm in diameter,
          ■ Commensal forms usually 15 to 20 μm
          ■ Invasive forms greater than 20 μm in greatest dimension
Entamoeba histolytica
●   Clinical diseases:
     ○ Amebic dysentery
           ■ Bloody diarrhea with abdominal cramping
           ■ Invasion of the intestinal mucosa occurs, producing ulceration
that may lead to perforation and peritonitis
     ○ Amebic colitis
           ■ Mimics ulcerative colitis and other forms of inflammatory
bowel disease
           ■ Symptoms: nonbloody diarrhea, constipation, abdominal
cramping, and weight loss.
     ○ Amebic liver abscesses
           ■ Most common form of extraintestinal amebiasis
           ■ Amebae are present in the stool in less than half of patients at
the time liver abscess is manifest.
           ■ Amebic hepatitis, characterized by an enlarged, tender liver in
someone with intestinal amebiasis, may occur in some cases
           ■ Amebomas form in response to the presence of amebae,
which in the intestine may cause Napkin ring lesion.
Nonpathologic Amebae
●   E. hartmanni
      ○ Called the small race of E. histolytica.
      ○ Cysts have a diameter of 10 μm and a singlenucleus
      ○ Trophozoites have a maximum diameter of 10- 12 μm
●   Entamoeba coli
      ○ Lumen-dwelling ameba
      ○ Compared to E. histolytica
           ■ More darkly cytoplasm
           ■ more vacuolated, containing numerous ingested bacteria,
yeasts, and other materials
           ■ Immature cysts have four nuclei that are larger
      ○ Mature cysts Contain eight nuclei and possibly 16 or more
Nonpathologic Amebae
●   Endolimax nana
       ○ Smallest ameba
       ○ Trophozoites often have atypical nuclei that contain a triangular
chromatin mass, a band of chromatin across the nucleus, or two
discrete masses of chromatin on opposite sides of the nuclear
membrane
●   I. bütschlii
       ○ Trophozoites and cysts have a large, centrally located
karyosome
            ■ Surrounded by achromatic granules
                   ● Appears as a muddy karyolymph space or halo
                   ● Halo is clear without evident achromatic granules Trichomonas vaginalis
                        ○ Indistinguishable from E. nana ●   Morphology
       ○ Cysts      ○ Resembles P. hominis but is larger (up to 23 μm)
            ■ contain a single nucleus           ■ The undulating membrane extends only half the length of the
            ■ karyosome is often eccentric with a nearby crescent of body
achromatic granules
            ■ Characterized by a prominent vacuole of glycogen that stains ●   Men
reddish brown in iodine-stained wet mounts      ○ Prostatitis or urethritis
Blastocystis hominis ●   Women
●   Inhabits the large bowel      ○ Common cause of vaginitis
      ○ Frequently found in stool specimens of asymptomatic individuals           ■ Inflammation, itching, vaginal discharge, and, occasionally,
●   Appears in stains as an ameba-like protozoan dysuria
●   Known to be a Stramenopile ●   Spread by sexual intercourse
●   Some studies have linked heavy infection to symptomatic intestinal      ○ Often by males who have an asymptomatic infection
disease, although this remains controversial.. ●   Diagnosis
      ○ Assumes one of three forms:      ○ Direct wet mount examination of
      ○ Vacuolated (seen most commonly)           ■ Vaginal fluid, prostatic fluid, or sediments of freshly passed
           ■ Usually spherical and variable in size (5 to 20 μm) urine
           ■ A central clear area and two to four peripheral nuclei           ■ May be insensitive (60% to 70% sensitivity for vaginal
      ○ Ameboid secretions)
           ■ Forms with bizarre shapes may predominate in heavy      ○ NAATs are now the recommended method for detection
infections Enteromonas hominis & Retortamonas intestinalis
      ○ Granular ●   Small
●   Nonpathogenic
FLAGELLATES ●   Intestinal flagellates that are seen infrequently but, when present,
Dientamoeba fragilis may occur in large numbers
●   Ameboid pathogen that infects the colon Balantidium coli
●   Symptoms:  ●   A ciliate
      ○ Diarrhea  ●   Acquired from hogs
      ○ Abdominal distention  ●   The largest protozoan
●   Spread by ingestion of pinworm eggs infected with D. fragilis  ●   The only ciliate to infect humans
●   Trophozoites  ●   Trophozoites
      ○ Contain two nuclei        ○ Between 40 μm and more than 200 μm largest (most measure
          ■ consist of a cluster of four to eight karyosomal granules 50 to 100 μm)
       ○ Uniformly covered with cilia that are slightly longer at the anterior
Giardia lamblia end adjacent to the cytostome
●   Also known as G. intestinalis and G. duodenalis        ○ Food vacuoles and contractile vacuoles present in the cytoplasm
●   Water-borne  ●   Cysts
●   Trophozoites        ○ Round
      ○ Multiplies in the small bowel        ○ Measuring 50 to 70 μm in length
      ○ Attaches to the mucosa by a ventral concave sucking disk  ●   Causes a dysentery-like syndrome with colonic ulcerations similar
      ○ Predominate in diarrheic stool to that of amebiasis
      ○ Pear-shaped with a tapered posterior end and two nuclei
●   Cysts COCCIDIA
      ○ Predominate in formed stools Cystoisospora belli
●   Infection may be asymptomatic ●   Undergoes both asexual and sexual development in the cytoplasm
      ○ May cause disease ranging from mild diarrhea with vague of small intestine epithelial cells
         abdominal complaints ●   Matures in passed stool
●   Diagnosis ●   Human infections cause diarrhea and malabsorption
      ○ Established by demonstration of trophozoites or cysts, or both, in       ○ Self-limited
         fecal specimens ●   In patients with AIDS or immunosuppressed
      ○ Direct wet mounts - “falling leaf” motility of trophozoites       ○ Persist for months or years, and may contribute to death
●   Diagnosis
Chilomastix mesnili       ○ Established by finding the unsporulated oocysts measuring
●   Nonpathogenic lumen-dwelling flagellate          12 × 30 μm in fecal specimens
●   Trophozoites       ○ Stained with acid-fast
      ○ Tapers of the end opposite the nucleus            ■ Red to reddish-orange
      ○ Three external flagella usually are not visible in stained or
         formalin fixed preparations Sarcocystis spp.
      ○ Single nucleus at one end of the organism ●   Acquired by ingestion of raw or incompletely cooked beef or pork
●   Cysts that contains cysts
      ○ Lemon-shaped ●   Usually is asymptomatic
      ○ Contain various curved cytostomal fibers with a safety pin–       ○ Occasionally diarrhea, abdominal pain, or anorexia
         like appearance ●   Sexual phase develops in the intestinal mucosa of carnivorous
animals
Pentatrichomonas hominis ●   Asexual, extraintestinal phase occurs in the muscles and tissues of
●   Known previously as Trichomonas hominis various intermediate hosts
●   Nonpathogenic intestinal flagellate ●   Depending on the spp., humans are intermediate/definitive hosts
●   Do not stain particularly well and often are distorted in ●   Diagnosis
permanent smears       ○ Best seen in wet mounts or in acid-fast–stained smears
●   Trophozoite       ○ Detection of sporulated 25 × 33 μm sporocysts in the stools
      ○ Single Entamoeba-like nucleus      Cryptosporidium spp.
      ○ Undulating membrane and associated costa, and flagella ●   Develops in the brush border of epithelial cells of the small and
      ○ Axostyle large intestine
          ■ A prominent rodlike object       ○ At times spreads to gallbladder, pancreas, and respiratory tract
          ■ Runs through the organism ●   A common cause of acute, self-limited diarrhea
          ■ Protrudes from the posterior end       ○ Especially children in day care
      ○ Lasts 9 to 23 days
●   In patients with AIDS
      ○ Chronic secretory diarrhea for months to years that contribute to
death
●   Diagnosis
      ○ Established by stool examination or antigen detection
      ○ Concentration methods, like formalin–ethyl acetate sedimentation
and Sheather’s sugar flotation
      ○ Acid-fast staining methods, like auramine - O or a modified cold
Kinyoun method
      ○ Commercial DFA and EIA reagents provide good sensitivity and
specificity
●   Spherical oocysts measure 4 to 6 μm in diameter when stained by
the modified Kinyoun, appears deep fuchsia
Cyclospora cayetanensis
●   Causes flu like illness with nausea, vomiting, weight loss, and
explosive watery diarrhea lasting 1 to 3 weeks
      ○ Diarrheal disease in both immunocompetent and
immunocompromised individuals
●  Described as a blue-green alga, a cyanobacterium-like body, or a
coccidian-like body
●   Oocysts
      ○ Passed unsporulated
      ○ Appear as non refractile spheres 8 to 10 μm in diameter
            ■ Contains a cluster of refractile globules enclosed within a
membrane under light microscopy
      ○ In trichrome-stained smears,
            ■ the oocysts appear as clear, round, and somewhat wrinkled
objects.

MICROSPORIDIA
 ●   Are obligate intracellular, spore-forming fungi
 ●   Multiplies intracellularly and form resistant spores eventually
rupturing the host cell
      ○ Infects adjacent cells
      ○ Passed out of the body
 ●   Spores
      ○ Contains a coiled polar tubule
           ■ Forcefully extruded under appropriate environmental stimuli
and penetrates the membrane of the recipient cell
           ■ Used to inject sporoplasm into host cell cytoplasm
 ●   Serious pathogens in immunocompromised hosts, especially with
AIDS
      ○ Cause 30% of otherwise unexplained diarrheal disease
      ○ Causes protracted diarrhea and weight loss
 ●   Diagnosis
      ○ Tissues under light or electron microscopy
      ○ Modified trichrome staining method for examination of stool
specimens for spores
           ■ (1.5 to 3 μm) elliptical spores stain red against a faint green
background
           ■ Some display a characteristic midbody cross-band