Lumen-

Dwelling
Protozoa
By:  Mintu,  MSc.    
 Taxonomic classification of Protozoans
Sub Phylum Sub-phylum Genus- Species-
kingdom examples examples

Protozoa Sarcomastig- Sarcodina-- - Entamoeba E. histolytica

ophora move by pseudopodia
further divided into

Mastigophora Giardia G. lamblia

move by flagella

Apicomplexa Plasmodium P. falciparum,

no organelle of P. vivax,
locomotion
P. malariae,
P. ovale

Ciliophora Balantidium B. coli

move by cillia

Microspora Enterocyto-zoa E. bienusi

Spore-forming
Examples of important intestinal protozoa
Transmitted by the faecal-oral route and
cause diarrhoea

•  Giardia lamblia: world-wide distribution,

lives in the small intestine and results in
malabsorption

Electron micrograph of G. lamblia

trophozoites (feeding stage)

•  Entamoeba histolytica: may invade the

colon and cause bloody diarrhoea –
amoebic dysentery. Also causes ameobic
liver abscess.

Typical flask-shaped ulcer due to E.

histolytica in the colonic mucosa
Examples of important intestinal protozoa
Transmitted by the faecal-oral route
and cause diarrhoea
•  Cryptosporidium parvum: more
prevalent in the
immunocompromised

•  Cyclospora cayatenensis -
parasitises the small intestinal
mucosa and may cause diarrhoea
for several weeks
Examples of important intestinal protozoa
Transmitted by the faecal-oral route
and cause diarrhoea

•  Balantidium coli: a large motile

ciliated parasite that lives in the
colon of pigs, humans and rodents
and can lead to colonic ulceration
Examples of important systemic protozoa
Detected in the blood

•  Plasmodium: the cause of malaria. There are 4

species that infect man: P. falciparum, P. vivax,
P. ovale and P. malariae

•  Toxoplasma gondi: transmitted by the ingestion

of oocysts from cat faeces. Infection can lead
to ocular problems and is also a cause of
neonatal toxoplasmosis

Typical lesion of •  Leishmania: transmitted by sand flies, can lead

cutaneous leishmaniasis
to visceral, cutaneous and mucocutaneous
leishmaniasis

Tsetse fly – the •  Trypanosoma: haemoflagellates which cause

vector of African –  In Africa - sleeping sickness (transmitted by
trypanosomiasis the Tsetse fly)
It has a painful
bite!
–  In South America - Chagas disease
(transmitted by the Reduviid bug)
Genus Entamoeba
Amoeba/Amebae
—  Non-pathogens —  Pathogens
—  E. gingivalis —  E. histolytica
—  E. coli —  E. polecki
—  E. hartmani —  Endolimax nana
—  E. dispar —  Iodamoeba butschlii
—  E. moshkovskii
Medical Significance of Protozoa
 Entamoeba histolytica
—  Anaerobic parasitic protozoan
—  Predominantly infecting humans and
other primates, E. histolytica is
estimated to infect about 50 million
people worldwide.
—  Mammals such as dogs and cats can
become infected transiently, but are
not thought to contribute significantly
to transmission.
Habitat & Morpho
—  Entamoeba histolytica is a parasite and lives in the mucous and
sub-mucous layers of the large intestine of man.
—  It may occur in the liver and lungs.
—  Rarely it invades brain, spleen etc. causing ulcers.
—  The disease caused by the parasite is known as “Amoebic
dysentery.”
—  It occurs in two forms—Trophic and Cystic.
 Entamoeba histolytica

Trophic form  

Pathognomonic/
Ingested RBC; distinctive nucleus  
Diagnostic Feature  

Cystic form  

Chromatoidal Body   'Cigar' shaped bodies (made up of crystalline ribosomes)  

Number of Nuclei   1 in early stages, 4 when mature  

Pathognomonic/
'Ring and dot' nucleus and chromatoid bodies  
Diagnostic Feature  
E. dispar and E. moshkkovskii
 Transmission
—  The active (trophozoite) stage exists only in the host and in fresh
loose feces; cysts survive outside the host in water, soils and on
foods, especially under moist conditions on the latter.
—  The cysts are readily killed by heat and by freezing temperatures, and
survive for only a few months outside of the host.
—  When cysts are swallowed they cause infections by excysting
(releasing the trophozoite stage) in the digestive tract.
—  The trophozoite stage is readily killed in the environment and cannot
survive passage through the acidic stomach to cause infection.
Pathogenesis
— E. histolytica, as its name suggests (histo–lytic
= tissue destroying), is pathogenic; infection
can lead to amoebic dysentery or amoebic
liver abscess.
— Symptoms can include fulminating
dysentery, bloody diarrhea, weight loss,
fatigue, abdominal pain, and amoeboma.
Pathogenesis
—  The amoeba can actually 'bore' into the intestinal wall,
causing lesions and intestinal symptoms, and it may reach
the blood stream.
—  From there, it can reach different vital organs of the
human body, usually the liver, but sometimes the lungs,
brain, spleen, etc.
—  A common outcome of this invasion of tissues is a liver
abscess, which can be fatal if untreated.
—  Ingested red blood cells are sometimes seen in the
amoeba cell cytoplasm.
 Tx
•  Metronidazole for the invasive trophozoites
PLUS a lumenal amoebicide for those still in
the intestine.
•  Paromomycin (Humatin) is the lumenal drug
of choice, since Diloxanide furoate
(Furamide) is not commercially available in
the USA or Canada (only being available
from the Centers for Disease Control and
Prevention).
•  A direct comparison of efficacy showed that
Paromomycin had a higher cure rate.

1.  Paromomycin (Humatin) should be used

with caution in patients with colitis as it is
both nephrotoxic and ototoxic.
2.  Absorption through the damaged wall of the
intestinal tract can result in permanent
hearing loss and kidney damage.
Recommended dosage:
—  Metronidazole 750mg tid orally, for 5 to 10 days
FOLLOWED BY Paromomycin 30mg/kg/day
orally in 3 equal doses for 5 to 10 days or
—  Diloxanide furoate 500mg tid orally for 10 days,
to eradicate lumenal amoebae and prevent relapse
E. coli
Endolimax nana
Note: tremendeous nuclear variation can mimic E. hartmanni, D. fragilis and
I. butschlii
Note: tremendous nuclear variation can mimic
E. hartmanni, D. fragilis and I. butschlii
Pathogenesis
—  Diarrhea and
pruritus
—  Occur in co-
infection with
Enterobius
vermicularis
(pinworm)
Opportunistic
free-living
amoeba
By:  Mintu,  MSc.  /  Masangkay,    RM  T  
                 
 General characteristics
 -­‐  inhabitants  of  soil,  water,  air-­‐conditioner  systems,  
humidifiers,  cooling  towers,  dust,  air,  compost,  
vegetation,  and  other  environmental  substrates.  
 
-­‐feeding  on  other  microscopic  organisms,  especially  
bacteria  and  yeast.  
 
-­‐associated  with  opportunistic  infection  of  the  CNS  and  
Acanthamoeba  causes  keratitis.  
 
Naegleria  fowleri  
 
 Trophozoite:  10-­‐35  um  flask  shaped  with  large  central  
karyosome,  uses  a  single  broad  pseudopod  for  
movement    
(if  exposed  to  warm  distilled  water  will  convert  to  
flagellated  form  within  1-­‐2  hours)  
 

Staining:  H&E    Fluorescent      Trichome  

  Morphology:  
 
 Cyst:  7-­‐15  um  in  diameter,  rounded,  thick-­‐walled,  
uninucleate    
(resistant  to  temperature  extremes  and  mild  chlorination)  
 

Trophozoite                    cyst  
  Mode  of  Infection:  
-­‐Swimming/diving  in  warm  ,  fresh  water  lakes,  rivers,  pods  and  pools  
 
Portal  of  entry:  
-­‐Enters  the  cribriform  plate  and  olfactory  bulbs  
   
 Portal  of  migration:  
-­‐Frontal  lobe  of  brain  
 
  Habitat:  
-­‐Warm,  fresh  waterlakes,  ponds,  pools,  rivers,  and  stagnant  waters  
 
Infective  stage:    Trophozoite  
 

 
 Virulence:    
q -­‐The  pathogenicity  of  Naegleria  amoebae  in  vivo  correlates  
with  resistance  to  complement-­‐mediated  lysis  in  vitro.  
q Nonpathogenic  Naegleria  species  activate  the  complement  
system  and  are  readily  lysed  by  complement.    
v In  contrast,  highly  pathogenic  N.  fowleri  activate  the  
complement  pathway  but  are  resistant  to  lysis.    
v The  ability  of  pathogenic  amoebae  to  escape  lysis  by  
complement  may  be  an  important  virulence  factor  in  the  
pathogenesis  of  primary  amoebic  meningoencephalitis.  
 
   Naegleria  fowleri  
 

 Pathology:  
-­‐Primary  amoebic  
meningoencephalitis  (PAM)  
-­‐acute  hemorrhagic  
meningoencephalitis  
(usually  fatal  within  1  week  
of  onset  of  symptoms)  
 
  Signs  and  symptoms:

•  severe  frontal  headache,    

•  fever,    
•  blocked  nose,    
•  altered  sense  of  taste  and  smell,    
•  stiff  neck,  and    
•  Kernig’s  sign    
 Diagnosis
u  -­‐CSF  
u  characteristic  predominance  of  
neutrophils,  no  bacterial  growth  on  
culture,  protein  moderately  
elevated,  glucose  moderately  
reduced.  

Note:  if  diagnosis  is  suspected,  CSF  should  not  be  

centrifuged  or  refrigerated  and  motile  amoebas  
can  often  be  detected  in  room  temperature  
 
  Diagnosis:      
 
-­‐Post-­‐mortem  autopsy  of  tissue/brain  sections  
(identification  of  trophozoites;  cysts  are  rarely  seen)  
 
-­‐Ante-­‐mortem  diagnosis  (identifying  trophozoites  in  CSF  
or  direct  mount,  stained  preparation)  
 
 
 
 
 
 
    
-­‐culture    
Ø  non-­‐nutrient  agar  plates:  1.5  %  agar,    
Ø  0.5  %  NaCl,    
Ø  pH  6.6-­‐7.0,    
Ø  seeded  with  a  lawn  of  heat  killed  or  living  Escherichia  
coli.    
Ø  Amoeba  ingest  the  bacteria,  leaving  tracks  in  the  
bacterial  lawn  seen  under  LPO)  
 
Subject:  intracerebral  inoculation  of  mice  
 
 
 
 
 
Treatment:  
 
ü  -­‐Amphotericin  B,    
ü  Sulfadiazine,    
ü  tetracycline  in  combination  
  Prevention:  
-­‐Because  of  the  wide  distribution  of  the  amoeba  
and  its  hardiness,  no  general  means  of  control  
exists.    
Public  swimming  pools  and  baths  must  be  
adequately  chlorinated  and  checked  periodically  
for  the  amoeba.  
 
   Acanthamoeba  sp.  
 

     
-­‐A.  culbertsoni  
-­‐A.  castellani  
-­‐A.  polyphaga  
-­‐A.  stronyxis  
 
A.  stronyxis          A.  castellani                A.  polyphaga  
  Morphology:  
 
Trophozoite:  
   

o   15-­‐45  um,    
o  display  needle-­‐like  filamentous  projections  
from  cell  known  as  acanthopodia  (irregularly  
spiculated  surface)  

SEM                        H&E  
  Morphology:  
 
 Cyst:    
 

o  10-­‐25  um,    
o  double-­‐walled  with  wrinkled  outer  wall  
(ectocyst)  and  polygonal,    
o  stellate  or  round  inner  wall  (endocyst)  
-­‐  

SEM                        H&E  
  Habitat:  
-­‐  Warm,  fresh  waterlakes,  ponds,  pools,  rivers,  and  
stagnant  waters  
 
 Infective  stage:  -­‐Trophozoite  
 
Mode  of  Infection:  
•  -­‐persons  who  use  daily-­‐wear  or  extended-­‐wear  soft  
contact  lenses  or  who  have  experienced  trauma  to  
the  cornea  
•  -­‐exposure  to  fresh  water  is  not  necessary  because  
cysts  readily  become  airborne  and  may  be  recovered  
from  throat  and  nasal  passages  
   
 Portal  of  entry:  
 
-­‐invasion  of  broken  skin,  conjunctiva,  
occasionally  the  lungs,  and  urogenital  epithelia  
 

Portal  of  migration:  

 
-­‐hematogenous  spread  from  primary  foci  in  
skin,  pharynx,  or  respiratory  tract  
 
   Pathology  
 
ü  -­‐Granulomatous  amoebic  encephalitis  (GAE)  

ü  -­‐Acanthamoeba  keratitis  

ü  -­‐Subacute  or  chronic  opportunistic  infection  of  

chronically  ill,  debilitated,  and  immunosupressed  
individuals,  leading  to  death  weeks  to  months  
following  onset  of  symptoms  

ü  -­‐Systemic  infections  occur  in  individuals  with  AIDS,  

and  may  present  as  ulcerative  skin  lesions,  
subcutaneous  abscesses,  or  erythematous  nodules  
 
   Acanthamoeba  sp.  
 

Subacute  or  chronic  opportunistic  

infection  of  chronically  ill
Acanthamoeba  keratitis  
•  -­‐ Development  of  
paracentral  ring  
infiltrate  of  the  
corneal  stroma,  which  
progresses  to  
ulceration  and  
possible  perforation,  
with  loss  of  the  eye  
 
Acanthamoeba  keratitis  
ü  -­‐recurrent  breakdown  and  healing  of  the  
overlying  epithelium  
ü  -­‐meningeal  signs,  alteration  of  mental  states,  
and  neurological  deficits  mimicking  brain  
abscess  or  tumor  
  Diagnosis:  
-­‐Demonstrating  trophozoites  or  cyst  in  corneal  scrapings  
or  biopsies  [stain:  Giemsa,  PAS,  Trichome,  fluorochrome  
Calcofluor  white  (cyst)]  
 
-­‐  

Indirect  
immunofluorescence  
Corneal  scraping            tissue  biopsy  
assay  
  
-­‐ Autopsy  or  brain  
biopsy:    
à Viable  tissues  
(trophozoite);    
à necrotic  tissues   Corneal  scraping                      tissue  biopsy  

(cysts)    
 
 

Indirect  
immunofluorescence  
assay  
-­‐ immunofluorescence  
and    
immunoperoxidase  
technique  (CDC)  
 
-­‐identification  to  the   tissue  biopsy  

species  level  is  

problematic  and  
reflects  uncertainty  as  
to  the  validity  of  the  
18  or  more  described  
species   Indirect  immunofluorescence  assay  
 
  Prevention:  
ü  -­‐not  swimming  and  diving  in  small  warm  fresh  
water  lakes  and  ponds  

ü  -­‐adequate  chlorination  of  swimming  pools  

ü  -­‐uses  of  contact  lenses  should  adhere  closely  to  

recommended  procedures  for  use  and  care  of  
lenses  
   Hartmannella  
 
   
 Pathology:  
   
respiratory  tract  infections  or  
ü  -­‐

generalized  fatal  meningoencephalitis  

ü  -­‐non-­‐pathogenic,  free-­‐living  amoeba  

 Infective  stage:  
-­‐Trophozoite  
 
  Morphology:  
   
   Trophozoite:    
ü  30  µm  long  and  6  µm  wide,  
monopodial  ("single  foot")  
ü  Possesses  filamentous  
speudopods  
ü  -­‐sluggish  motility  
ü  -­‐characteristic  walled  cyst  
that  may  contain  pores  
ü  -­‐non-­‐flagellates  in  water  
ü  -­‐do  not  encyst  in  tissue  

 Habitat:  
-­‐  Warm,  fresh  water  lakes,  ponds,  pools,  
rivers,  and  stagnant  waters  
 
 Balamuthia  mandrillaris  
   

 Pathology:      
-­‐granulomatous  amoebic  encephalitis  (GAE)  
 

Signs  and  symptoms:  

-­‐focal  paralysis,  seizures,  
and  brainstem  
symptoms  such  as  facial  
paralysis,  difficulty  
swallowing,  and  double  
vision.  
   
  Morphology:  
 

Brain  tissue:  H&E  staining  

 Trophozoite:  
     15-­‐60  µm  (pleomorphic),  produce  long,  slender  

pseudopodia,  contain  a  large  nucleus  with  a  large,  

centrally-­‐located  karyosome  but  no  peripheral  
chromatin,    there  is  no  flagellated  trophozoite  stage  as  
in  Naegleria  spp.  
 
  Morphology:  
 
 Cyst:    
•  10-­‐25  µm  in  diameter,  have  two  walls:  a  wrinkled  
fibrous  outer  wall  (exocyst)  and  an  inner  wall  
(endocyst)  that  may  be  hexagonal,  spherical,  star-­‐
shaped  or  polygonal.      
•  Cysts  contain  only  one  nucleus  with  a  large  
karyosome.  may  be  found  in  the  brain,  eyes,  skin,  
lungs  and  other  organs  
 

Brain  tissue:  H&E  staining  

  Habitat:  
•  -­‐Balamuthia  has  not  been  definitively  isolated  in  
nature,  but  it  is  believed  to  be  distributed  throughout  
the  temperate  regions  of  the  world.    
•  This  is  supported  somewhat  by  the  presence  of  
antibodies  to  Balamuthia  present  in  healthy  
individuals.  
 

     

Infective  stage:  
-­‐Trophozoite  &  cyst  
 
  Portal  of  entry:  
-­‐lower  respiratory  tract  or  through  open  wounds  

Portal  of  migration:  

 

•  -­‐amoebas  may  form  a  skin  lesion  or  migrate  

to  the  brain  
•  -­‐may  enter  the  bloodstream  and  migrate  to  
the  brain  
 
  Diagnosis:  
-­‐Trophozoite  and  cyst  inclusions  in  the  brain  tissue  on  
microscopic  examination  of  brain  biopsies  performed  
on  infected  individuals.  
 
-­‐monoclonal  or  polyclonal  antisera  in  
immunofluorescence  or  immunoperoxidase  assay  
 
-­‐does  not  grow  on  agar  plates  like  Naegleria  and  
Acanthamoeba,  cannot  be  cultured  on  an  agar  plate  
coated  with  gram-­‐negative  bacteria  because  unlike  
most  amoeba,  Balamuthia  mandrillaris  does  not  feed  on  
bacteria  
 
B.  mandrillaris  pleomorphic  trophozoite  

B.  mandrillaris  cyst  in  brain  biopsy  H&E    

 References:  
 

Henry’s  Clinical  Diagnosis  and  

Management  by  Laboratory  Methods,  
21st  ed.,  2007  
A  Manual  of  Laboratory  Diagnostic  
Tests,  7th  ed.,  F.  Fischback,  2004  
Microbiology  a  Systems  Approach,  M.  
Cowan,  K.  Talaro,  2006  
Introduction  to  Diagnostic  
Microbiology,  M.  Delost,  1997  
Basic  Clinical  Parasitology,  F.  Neva,  H.  
Brown,  1994  
 
GOOD
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