1. Protozoa (原虫)

2. General Account
One-cell animal – monocellular or unicellular organisms with full vital functions
Species – total named species:65,000; parasitic: around 10,000

3. Classification of protozoa
Amoebae
Flagellates
Sporozoa
Ciliates

4. Life cycle patterns One-host form Two-host form
One stage form – Trophozoite
Two stage form – Trophozoite & Cyst
Two-host form
Mammals mammals
Mammals insect vectors

5. Mode of Reproduction Asexual Reproduction Sexual Reproduction
Binary fission – result in 2 daughter cells
Schizogony – multiple fission result in multiple cells
Budding
Exogenous budding - by external budding result in multi- cells
Endodyogony - by internal budding result in 2 cells
Sexual Reproduction
Conjugation – exchange of nuclear material of 2
Gametogony – sexually differentiated cells unite -- zygote

6. Opportunistic & Accidental (protozoa) infections
Pathogenesis
Opportunistic & Accidental
(protozoa) infections
Host Resistance
Innate immunity
Acquired immunity
Parasite Invasion
Toxin
Mechanically damage
Immune impair
Immune inhibition
hypersentivity

7. Opportunistic parasites
Opportunistic infection
An infection by a microorganism that normally does not cause disease but becomes pathogenic when the body's immune system is impaired and unable to fight off infection

8. Amoebic Infections
Entamoeba histolytica
Acanthamoeba
Naegleria

9. Epidemiology
4th leading cause of death from parasitic diseases worldwide
Organism # of deaths/yr # infected
Entamoeba ~75,000 ~300 million
Ascaris ~200, ~480 million
Schistosoma ~750,000 ~200 million
Plasmodium 2-3 million ~500 million
(Malaria)
Amoebiasis is not restricted to the tropics and subtropics, it also occurs in temperate and even in arctic and antarctic zones

10. Contaminated water is a source of infection.

11. Infection is common in developing countries where sanitation is poor.

12. Amoeba in alimentary tract
Entamoeba
E. histolytica (pathogenic)
E. dispar (non-pathogenic)
E. coli (big sister)
E. hartmani (little brother)
E. gingivalis (oral)
Endolimax nana (occasionally pathogenic)
Iodamoeba butschlii

13. Morphology

14. Morphology E. histolytica trophozoite Ingested RBC Endoplasma
Ectoplasma
Nucleus with central karyosome and finely divided chromatin granules
Pseudopod
E. histolytica trophozoite

15. Trophozoites Morphology Single nucleus with a central,
dot-like karyosome

16. Micrograph of a trophozoite ingesting a red blood cell deprived from its host.

17. Morphology
1-4 ring-like nuclei with finely divided peripheral chromatin
Cyst wall and round shape
Mature E. histolytica Cyst

18. Morphology

19. Morphology
E. Coli trophozoites

20. E. Coli cysts
Morphology

21. E. histolytica Stages - CYSTS
Infective Stage for humans
Resistant walls maintain viability
If moist can last several weeks
Killed by desiccation or boiling
Diagnostic Stage in formed stools
Can be concentrated and stained easily
Not seen in liquid (diarrheic) stools or tissues

22. E. histolytica Stages - TROPHOZOITES
Cause amoebiasis (damage tissue)
Spread throughout the body, but ...
Rarely transmit the infection to others
Labile in liquid stools or tissue, and
must be rapidly found or preserved (quick fixation & cold storage) for Diagnosis

23. Life cycle

24. Life cycle Humans acquire E. histolytica by:
Ingesting cysts (4 nuclei mature) in fecally contaminated food or water
Rarely by directly inoculating trophozoites into colon or other sites
(anal sex?)
Fecal-Oral transmission (hand to mouth)

25. Life cycle
The basic generation-cycle: cyst – lumen trophozoites – cyst
Trophozoites may invade intestine and spread
Cyst formation – essential factors: enviroment + time
Infective cysts and trophozoites pass in feces

26. Pathogenesis General Types of Virulence Factors: Adherence factors
260kDa Gal/GalNAc lectin
Invasion factors
Amoeba pores
Cysteine proteinases
Endotoxins

27. Pathogenesis Trophozoites ... Attach to mucosal epithelial cells (MEC)
Lyse MEC
Ulcerate and invade mucosa
Cause dysentery (diarrhea + blood)
Metastasize via blood &/or lymph to
Form abscesses in extraintestinal sites ...

28. Clinical Classification of Amoebiasis (World Health Organization)
Asymptomatic Infection:"Cyst Passers/carrier”
Symptomatic Infection:
Intestinal Amoebiasis: (colon and rectum盲肠、 升结肠、直肠、乙状结肠和阑尾)
Acute Dysenteric (dysentery)
Chronic Non-Dysenteric (“self-cured”)
Extra-Intestinal Amoebiasis:
Amoebic Liver Abscess (ALA)
Amoebic Pulmonary Abscess
Other sites (brain, skin, GU, ?)

29. Clinical classification
Asymptomatic infection (carrier)  >90% (E. dispar?)
Symptomatic cases <10%
8% -10% dysentery, colitis, etc
2% invasive amoebiasis
0.1% deaths

30. Acute Dysenteric Amoebiasis
Clinical manifestation
Symptoms:
Bloody mucoid diarrhea
RBCs and few WBCs in stools
Abdominal pain
weight loss
bloating, tenesmus(里急后重) and cramps

31. Acute Dysenteric Amoebiasis
Clinical manifestation
Signs:
Fever (33%)
Tender (enlarged) liver
Stools positive for trophozoites +/- WBC
NO cyst in loose stools

32. Clinical manifestation
Pinpoint lesion on mucous membrane
Flask-shaped crateriform ulcers
Pathological changes in large intestine

34. Chronic Non-Dysenteric Amoebiasis
Clinical manifestation
Chronic Non-Dysenteric Amoebiasis
“self-cured” carrier state
Usually for 1 year, 37% symptomatic >5 years
Intermittent diarrhea, mucus, abdominal pain, flatulence and/or weight loss
E. histolytica trophs in loose stools
Cysts in solid stools
Positive serology and ulcerations on sigmoidoscopy or pathologic test

35. Amoebic Liver Abscess (ALA)
Clinical manifestation
Extra-Intestinal Amoebiasis
Amoebic Liver Abscess (ALA)
Symptoms
History of dysentery (1 yr), weight loss, abdominal pain, chest or shoulder pain
Signs
fever, hepatomegaly
Diagnostic aspiration:non-odorous, reddish-brown in color aspirate (chocolate jam) "anchovy paste"
Might find trophozoites in the aspirate
Skin inflammation

36. Clinical manifestation
Ulcers caused by invasion of E. histolytica into the liver.

37. Clinical manifestation

38. Clinical manifestation

39. An Amoebic Liver Abscess Being Aspirated.
Note the reddish brown color of the pus (‘anchovy-sauce’). This color is due to the breakdown of liver cells.
Gross pathology of amoebic abscess of liver. Tube of "chocolate" pus from abscess. 

41. X-ray of Amoebic Liver Abscess
Clinical manifestation

42. Diagnosis Pathogenic diagnosis
Stool examination:
Direct Fecal Smear (trophs and cysts)
Fecal concentration and iodine dye techniques - (cysts) ZnSO4 or formalin-ether
Cultivation
DNA detection
Sigmoidoscopy
Serologic Tests (for chronic disease): ELISA, IHA (indirect hemagglutination)
Imaging: X-ray; CT

43. Stool examination trophozoite cyst loose feces solid feces
specimen
loose feces
solid feces
method
direct smear with normal saline
direct smear with iodine stain
diseases
amoebic dysentery
chronic intestinal amoebiasis or carriers
remarks
1.container must clean 2.examined soon after they have been passed. 3.select bloody and mucous portion.

44. Two microscopically indistinguishable Entamoeba sp.
E. histolytica
invades tissues
should always be treated
E. dispar
is non-pathogenic, even in AIDS
should not be treated

45. Treatment of Amoebiasis
For invasive forms: metronidazole
For luminal forms: Iodoquinofonum, paromomycin, diloxanide
Do not treat asymptomatic intestinal E. dispar infection

46. Treatment of Amoebiasis

47. Prevention & Control Individual measures Chemotherapeutic Trial
Diagnosis and treatment of E. histolytica patients
Safe drinking water (boiling or 0.22 µm filtration)
Cleaning of uncooked fruits and vegetables
Prevention of contamination of foods
Chemotherapeutic Trial

48. Community measures Prevention & Control Public services and utilities
Adequate disposal of human stools
Safe and adequate water supply
Primary health care systems
Health education (washing hands, cleaning and protecting food, controlling insects)
Specific surveillance programs and Control programs integrated into ongoing sanitation & diarrhea control
Health Regulations
Control of food vendors and food handlers
Control of flies and cockroaches

49. Infections with Free Living Amoebae
Naegleria 耐格里属
Acanthamoeba 棘阿米巴属

50. Free Living Amoebae Not seen in humans Naegleria
10-35 µm (smaller than A. spp.) with lobate pseudopodia
Acanthamoeba cysts & trophs are seen in humans i
15-45 µm with filiform pseudopodia

51. Acanthamoeba spp.
Acanthamoeba trophozoites with acanthopodia

52. Primary Amoebic Meningoencephalitis PAME
An acute suppurative infection of the brain and meninges that is rapidly fatal and usually not diagnosed antemortem
Caused by Naegleria fowleri
Headache, lethargy and olfactory problems
Sore throat, runny nose, severe headache, vomiting, stiff neck, confusion leading to ...
Coma and death

53. DIAGNOSIS Patient History (child) Symptoms/Signs PAME
Prior Health Excellent
Recent History of Swimming (fresh water/pools)
Cases peak during HOT months
Symptoms/Signs
Sore throat, runny nose, headache, vomiting, stiff neck, mental confusion, olfactory problems, lethargy, coma and death

54. Treatment PAME None effective - few patients survive
Amphoteracin B +/- ?

55. Granulomatous Amoebic Encephalitis GAE
A more slowly progressive, chronic form of the disease not associated with swimming (except in hot tubs)
cause: Acanthamoeba castellanii
history of subcutaneous nodules, eye or skin infection, progressive nasal congestion, headache ...
CNS lesions with negative serology for toxoplasmosis
in debilitated/immuno-compromised Pts with CD4+ TL <200/mm3
disseminated infection: skin, sinuses, lungs, CNS/CSF

56. Pathology abscesses/lesions (tissues) have amoebae rarely seen in CSF
GAE
Pathology
abscesses/lesions (tissues) have
granulomatous inflammation
hemorrhagic necrosis and vasculitis
trophozoites & cysts with wrinkled-walls!
amoebae rarely seen in CSF

57. GAE
Treatment
No satisfactory or effective treatment ?
amphotericin B

58. Acanthamoeba Keratitis AK
Corneal infection with Acanthamoeba spp. trophozoites & cysts
Ulcerations & “Ring Infiltrate” of cornea
Induced by
trauma to eye, exposure to contaminated H2O
contact lens wear with tap water rinsing

59. AK Diagnosis Treatment Examine corneal scrapings or smear
Histopathologic examination of cornea
Treatment
Triple Antiamoebic Therapy
neomycin-polymyxin-gramicidin/propamidine/miconazole
Penetrating keratoplasty (cadaver cornea)

61. Plasmodium (疟原虫)

62. History
Malaria is an old infectious disease. The first documentation about it is at 1500BC.
Until the end of the 19th century, it was commonly thought that malaria was caused by breathing bad air (mal-aria) and was associated with swamps

63. History
Important application of the knowledge about malaria: W. Gorgas successfully implemented control strategies for malaria and yellow fever during the construction of Panama Canal

66. Global distribution

67. Plasmodium that infect human

68. Malaria current status

69. Morphology
Plasmodium is the one-cell parasite, so the basic morphology is a nucleus (chromatin), cytoplasma and cell membrane
Wright or Giemsa stain gives the Cytoplasm – bluish; Chromatin - red to red-purple while the malarial pigments are yellow-brown
There are three stages and six main forms of plasmodium in RBC

70. Plasmodium in RBC
Trophozoites (滋养体期)：ring form and developing trophozoites Schizonts (裂殖体期)：immature and mature -- merozoites Gametocyte (配子体期)： Microgametocytes and macrogametocytes

71. Trophozoites
Fig. 1: normal red cell; Figs. 2-5: ring stage parasites (young trophozoites)

72. Ring form trophozoites
Thin blood film (Giemsa stained)
Ring like plasma with one nucleus at one side

73. Mature trophozoites (amoeboid form)
The plasmodium grow with pseudopods, more cytoplasma and malarial  pigment presented in the plasma
Red blood cell enlarged and became pale with Schüffner‘s  dots(薛氏小点)

74. Schizonts
Figs.： increasingly mature schizonts

75. Macrogametocyte (female gametocyte) of P.v
Giemsa staining
compact nucleus, usually at edge of the parasite
scattered pigment granules
The gametocyte is completely filling its host cell

76. Microgametocyte (male gametocyte) of P.v
Giemsa staining
large nucleus at the center of the cell
scattered pigment granules

77. Macrogametocyte of P. f
The crescent-shaped gametocytes of P. falciparum are very distinctive, but tend to only appear late in the infection
Compact nucleus, red, usually at the center of the cell
Malarial pigments around the nucleus

78. Microgametocyte of P. f Sausage-shaped with two blunt end
Large nucleus at the center
Sometimes hard to distinguish from the female gametocytes

81. exo-erythrocytic stage—
merozoites in liver cells

82. The vector – female Anopheles

83. Development in the vector
Gametocytes zygote oocyst sporozoites

84. Life Cycle

85. Life cycle Infective stage：sporozoites Transmission
female Anopheles mosquito
transfusion
transplacental
needle stick
Pathogenic stages：erythrocytic stage
Diagnositic stages：erythrocytic stage

86. Pathogenesis

87. Clinical features Incubation period （潜伏期）
Time interval between the mosquito bite and the onset of the clinical symptoms
Time for the sporozoite reaching liver and entering
Duration of the development in the liver
Time of development in the RBC to produce sufficient erythrocytic merozoites to cause clinical symptoms
P. falciparum 7 to 27 days
P. vivax 8 to 31 days
P. malarie 18 to 40 days
P. ovale 16 to 18 days

88. Clinical features Typical malaria paroxysm（发作）
Malarial paroxysm -symptom for erythrocytic phase
RBCs rupture releasing merozoites, malarial metabolites, endotoxin in blood
shaking chill（寒战） followed by fever （高热）(2-20 hrs)
profuse sweating when fever breaks（出汗退热）
repeated characteristic cycle for each species
P. falciparum 36-48hrs
P. vivax 48hrs
P. malariae 72hrs
P. ovale 48hrs

89. Recrudescence versus relapse
Recrudescence （再燃） is the return of the symptom of malaria after apparent cure, which is due to a sudden increase in what was a persistent, low-level parasite population in the blood.
The periodic increase in numbers of parasites results from a residual population persisting at very low levels in the blood after inadequate or incomplete treatment of the initial infection.
The asymptomatic situation may last for as long as 53 years.
manifestation and proliferation of chemo-resistant cell clones in the cancer. This is considered acquired resistance. In same cases resistance to chemotherapy can occur even before the use of chemotherapy and is considered intrinsic resistance.

90. Recrudescence versus relapse
Victims may suffer relapse （复发）after apparent recovery of malaria. This is caused by the long prepatent sporozoites (LPPs) or hypnozoites which remain dormant in the hepatocytes for an indefinite period.
P. vivax and P. ovale will develop hypnozoites in the liver responsible for relapse while patients infected with P. falciparum and P. malariae have no phenomenon of relapse
All four types of plasmodium can maintain low level of parasitemia and account for the recrudescence of the disease

91. Clinical features liver & spleen damage of long-term
complications from P. falciparum
Anemia
tropical splenomegaly syndrome
cerebral malaria - 50% of deaths
coma and renal failure due to tubular necrosis

92. tropical splenomegaly syndrome

93. Cerebral malaria

94. Diagnosis
Travel history to endemic area & presence of symptoms, e.g., fever and chills
Thick smears - hard to read
Thin smears - see RBC morphology and parasite characteristics
serology - helps rule out fever of unknown origin
DNA probes- too slow & costly esp. in field

95. Diagnosis
Thick blood smear
Thin blood smear

96. Indirect fluorescent antibody test （IFA）

97. DNA--PCR
Lane S: molecular base pair standard (50-bp ladder).  Black arrows show the size of standard bands.
Lane 1: P. vivax (size: 120 bp)
Lane 2: the red arrow shows the P. malariae (size: 144 bp)
Lane 3: P. falciparum (size: 205 bp)
Lane 4: P. ovale (size: 800 bp)

98. Treatment
Quinidine, chloroquine, primaquine, pyrimethanime, sulfadoxine, mefloquine, tetracycline, proguanil
affect parasite in different ways depending on stage when administered
different species react differently
emergence of drug-resistant malaria

99. Prevention and control
Personal protection- netting, repellents, etc.
mosquito control or eradication - not easy
avoid sharing needles
develop vaccines - not currently available
select proper treatment for species and morphological forms