Amoebiasis is caused by the parasite Entamoeba histolytica. It infects the intestinal lining and can cause dysentery or amoebic colitis. In severe cases, it can lead to liver or brain abscesses. Diagnosis involves microscopic examination of stool samples to look for the motile trophozoites or cysts of E. histolytica. Specific diagnosis requires demonstrating the invasive trophozoites that may contain ingested red blood cells. Amoebiasis remains common in developing tropical areas with poor sanitation but can infect travelers as well.

1. AmoebiasisAmoebiasis
Dr.T.V.Rao MDDr.T.V.Rao MD

2. ENTAMOEBAENTAMOEBA
HISTOLYTICAHISTOLYTICA
 Entamoeba histolyticaEntamoeba histolytica
was first described bywas first described by
Lambl in 1859 and LoschLambl in 1859 and Losch
established it pathogenicestablished it pathogenic
nature in 1875 in anature in 1875 in a
dysenteric patient isdysenteric patient is
St.PetersbergSt.Petersberg
 Councilman and lafleur inCouncilman and lafleur in
1981 described amoebic1981 described amoebic
liver abscess.liver abscess.
 Schauudinn ( 1903 )Schauudinn ( 1903 )
differentiated pathogenicdifferentiated pathogenic
and nonpathogenic typesand nonpathogenic types
of Amoebaeof Amoebae

3. AmebiasisAmebiasis
 Amebiasis (am-e-BI-a-Amebiasis (am-e-BI-a-
sis) is a disease causedsis) is a disease caused
by a one-celled parasiteby a one-celled parasite
calledcalled EntamoebaEntamoeba
histolytica (ent-a-ME-bahistolytica (ent-a-ME-ba
his-to-LI-ti-ka)his-to-LI-ti-ka)..
Although it is moreAlthough it is more
common in people whocommon in people who
live in tropical areas withlive in tropical areas with
poor sanitary conditionspoor sanitary conditions

4. Amoebiasis a Major HealthAmoebiasis a Major Health
ProblemProblem
 Amoebiasis is estimated to cause 70,000Amoebiasis is estimated to cause 70,000
deaths per year world wide Symptoms candeaths per year world wide Symptoms can
range from mild diarrhea to dysentery withrange from mild diarrhea to dysentery with
blood and mucus in the stool.blood and mucus in the stool. E. histolyticaE. histolytica
is usually a commensals organism.is usually a commensals organism.
Severe Amoebiasis infections (known asSevere Amoebiasis infections (known as
invasive orinvasive or fulminantfulminant amoebiasis) occur inamoebiasis) occur in
two major forms. Invasion of the intestinaltwo major forms. Invasion of the intestinal
lining causes amoebic dysentery orlining causes amoebic dysentery or
amoebic colitis.amoebic colitis.

5. Trends of AmoebiasisTrends of Amoebiasis

6. Transmission of AmebiasisTransmission of Amebiasis
 Amoebiasis is transmittedAmoebiasis is transmitted
by fecal contamination ofby fecal contamination of
drinking water and foods,drinking water and foods,
but also by direct contactbut also by direct contact
with dirty hands orwith dirty hands or
objects as well as byobjects as well as by
sexual contact.sexual contact.
Additionally, geophagy isAdditionally, geophagy is
a common route ofa common route of
infection in certaininfection in certain
cultures.cultures.
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7. Nature of the diseaseNature of the disease
 Symptoms are usually gastrointestinal includingSymptoms are usually gastrointestinal including
diarrhoea, vomiting, abdominal pain ordiarrhoea, vomiting, abdominal pain or
discomfort and fever. Symptoms take from a fewdiscomfort and fever. Symptoms take from a few
days to a few weeks to develop and manifestdays to a few weeks to develop and manifest
themselves, but usually it is about two to fourthemselves, but usually it is about two to four
weeks. Most infected people are asymptomaticweeks. Most infected people are asymptomatic
but this disease has the potential to make thebut this disease has the potential to make the
sufferer dangerously ill, especially if there is anysufferer dangerously ill, especially if there is any
suggestion of immunocompromised.suggestion of immunocompromised.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

9. Events on AmoebiasisEvents on Amoebiasis

11. Trophozoites ofTrophozoites of
E.histolyticaE.histolytica

12. Trophozoites and CysticTrophozoites and Cystic
stagesstages

13. Cystic stage -Cystic stage -
E.histolyticaE.histolytica

15. Amoebiasis causes EpithelialAmoebiasis causes Epithelial
damagedamage

16. Numerous EosinophilicNumerous Eosinophilic
spherical structure withinspherical structure within
necrotic areanecrotic area..

17. Tissue showing AmoebicTissue showing Amoebic
infectioninfection
 The sphericalThe spherical
structurestructure
(Trophozoites) has(Trophozoites) has
one basophilicone basophilic nucleinuclei
about the size ofabout the size of
RBC’s. Note someRBC’s. Note some
RBC's areRBC's are
phagocytosed by thephagocytosed by the
TrophozoitesTrophozoites
(erythrophagocytosis)(erythrophagocytosis)
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18. Virulence factorsVirulence factors
 Trophozoites of E.histolytica interact with host through aTrophozoites of E.histolytica interact with host through a
series of stepsseries of steps
1 Adhesion of target cell, phagocytosis and cytopathic1 Adhesion of target cell, phagocytosis and cytopathic
effecteffect
2 E.histolytica induces both Humoral and cell mediated2 E.histolytica induces both Humoral and cell mediated
immune responses.immune responses.
3 Virulence factors – In many circumstances lumen3 Virulence factors – In many circumstances lumen
dwelling Amoeba may be asymptomaticdwelling Amoeba may be asymptomatic
4 Causes disease only when invade the Intestine4 Causes disease only when invade the Intestine
5 Virulence is associated with secretion of Cysteine5 Virulence is associated with secretion of Cysteine
proteniase which assists the organism in digesting theproteniase which assists the organism in digesting the
extracellular matrix and invading tissuesextracellular matrix and invading tissues

19. Cysteine proteinase -Cysteine proteinase -
Complement factorComplement factor C3C3
 It is observedIt is observed
Cysteine proteinaseCysteine proteinase
produced by invasiveproduced by invasive
strains of E.histolyticastrains of E.histolytica
inactivates theinactivates the
complement factorcomplement factor
C3C3 and are thusand are thus
resistant toresistant to
ComplementComplement
mediated lysis.mediated lysis.

20. Cysteine proteinase virulentCysteine proteinase virulent
factorfactor
 Cysteine proteinase is anCysteine proteinase is an
important virulent factorimportant virulent factor
 Its presence makesIts presence makes
E.histolytica is resistant toE.histolytica is resistant to
complement mediated lysiscomplement mediated lysis
 Can cleave the extracellularCan cleave the extracellular
structural matrix andstructural matrix and
degrade fibronectin anddegrade fibronectin and
laminin, as well as type Ilaminin, as well as type I
collagen.collagen.
 In this process basementIn this process basement
membrane is degraded andmembrane is degraded and
leads to invasionleads to invasion

21. ZymodemeZymodeme
 Lectin bindingLectin binding
Zymodeme analysis,Zymodeme analysis,
genome specific DNAgenome specific DNA
analysis and staininganalysis and staining
with Monoclonalwith Monoclonal
antibodies have beenantibodies have been
successfully used assuccessfully used as
markers to identifymarkers to identify
invasive strains ofinvasive strains of
E.histolyticaE.histolytica

22. Types of ZymodemesTypes of Zymodemes
 Based onBased on
ElectrophoreticElectrophoretic
mobility E.histolyticamobility E.histolytica
strains are classifiedstrains are classified
into 22 Zymodemesinto 22 Zymodemes
 However only 9 areHowever only 9 are
invasiveinvasive

23. Invasive x NoninvasiveInvasive x Noninvasive
strainsstrains
 The invasive and nonThe invasive and non
invasive strains mayinvasive strains may
appear identical mayappear identical may
represent two distinctrepresent two distinct
speciesspecies
 11 Invasive strain –Invasive strain –
E.histolyticaE.histolytica
 22 Non invasive strainsNon invasive strains
reclassified asreclassified as
E.dispar.E.dispar.
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24. Host Factor ContributionsHost Factor Contributions
 Several factors contribute to influenceSeveral factors contribute to influence
infectioninfection
1 Stress1 Stress
2 Malnutrition2 Malnutrition
3 Alcoholism3 Alcoholism
4 Corticosteriod therapy4 Corticosteriod therapy
5 Immunodeficiency5 Immunodeficiency
6 Alternation of Bacterial flora6 Alternation of Bacterial flora
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

25. Risk FactorsRisk Factors
 People in developing countries that havePeople in developing countries that have
poor sanitary conditionspoor sanitary conditions
 Immigrants from developing countriesImmigrants from developing countries
 Travellers to developing countriesTravellers to developing countries
 People who live in institutions that havePeople who live in institutions that have
poor sanitary conditionspoor sanitary conditions
 HIV-positive patientsHIV-positive patients
 Men who have sex with menMen who have sex with men
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26.  No symptoms (in theNo symptoms (in the
majority of cases),majority of cases),
 VagueVague
gastrointestinalgastrointestinal
distress,distress,
 Dysentery (with bloodDysentery (with blood
and mucus).and mucus).
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series
DysenteryDysentery

27. How the Amebiasis ManifestsHow the Amebiasis Manifests
 Most cases of amebiasis have very mildMost cases of amebiasis have very mild
symptoms or none.symptoms or none.
 More severe infection may cause fever,More severe infection may cause fever,
profuse diarrhea, abdominal pain,profuse diarrhea, abdominal pain,
jaundice, anorexia, and weight loss.jaundice, anorexia, and weight loss.
 In severe cases, it can lead toIn severe cases, it can lead to
development of abscesses (pockets ofdevelopment of abscesses (pockets of
amoebae and inflammatory cells) in theamoebae and inflammatory cells) in the
liver or, more rarely, the brain.liver or, more rarely, the brain.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

28. Clinical symptoms are VagueClinical symptoms are Vague
 Wide spectrum, from asymptomaticWide spectrum, from asymptomatic
infection ("luminal amebiasis"), to invasiveinfection ("luminal amebiasis"), to invasive
intestinal amebiasis (dysentery, colitis,intestinal amebiasis (dysentery, colitis,
appendicitis, toxic mega colon,appendicitis, toxic mega colon,
amebomas), to invasive extra intestinalamebomas), to invasive extra intestinal
amebiasis (liver abscess, peritonitis,amebiasis (liver abscess, peritonitis,
pleuropulmonary abscess, cutaneous andpleuropulmonary abscess, cutaneous and
genital amoebic lesions).genital amoebic lesions).
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

29. Diagnosis ofDiagnosis of
AmebiasisAmebiasis

30. BASICS METHODS INBASICS METHODS IN
DIAGNOSISDIAGNOSIS
 Fresh stool: wet mounts andFresh stool: wet mounts and
permanently stained preparations (e.g.,permanently stained preparations (e.g.,
trichrome).trichrome).
 Concentrates from fresh stool: wetConcentrates from fresh stool: wet
mounts, with or without iodine stain, andmounts, with or without iodine stain, and
permanently stained preparations (e.g.,permanently stained preparations (e.g.,
trichrome). Concentration procedures,trichrome). Concentration procedures,
however, are not useful forhowever, are not useful for
demonstrating Trophozoites.demonstrating Trophozoites.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

31. DiagnosisDiagnosis of Amebiasisof Amebiasis
 Diagnosis of amebiasis can be very difficult. OneDiagnosis of amebiasis can be very difficult. One
problem is that other parasites and cells canproblem is that other parasites and cells can
look very similar tolook very similar to E. histolyticaE. histolytica when seenwhen seen
under a microscope. Therefore, sometimesunder a microscope. Therefore, sometimes
people are told that they are infected withpeople are told that they are infected with E.E.
histolyticahistolytica even though they are not.even though they are not. EntamoebaEntamoeba
histolyticahistolytica and another ameba,and another ameba, EntamoebaEntamoeba
dispardispar, which is about 10 times more common,, which is about 10 times more common,
look the same when seen under a microscopelook the same when seen under a microscope
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

32. MicroscopyMicroscopy
 This is the traditionalThis is the traditional
means of diagnosing themeans of diagnosing the
disease—one simplydisease—one simply
looks at a sample of stoollooks at a sample of stool
under a microscope.under a microscope.
BecauseBecause E. histolyticaE. histolytica isis
not always found in everynot always found in every
stool sample, severalstool sample, several
samples from differentsamples from different
days may be needed.days may be needed.
Sometimes red bloodSometimes red blood
cells that have beencells that have been
ingested by the parasiteingested by the parasite
are visible.are visible.

33. Microscopic examination ofMicroscopic examination of
StoolStool
 A sample of freshlyA sample of freshly
collected fecalcollected fecal
specimen containingspecimen containing
mucous and blood ismucous and blood is
transferred on atransferred on a
slightly warm slideslightly warm slide
and covered withand covered with
cover slip andcover slip and
examinedexamined
microscopicallymicroscopically
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

34. EE. histolytica. histolytica //E. disparE. dispar
cystcyst..

35. E. histolyticaE. histolytica//E. disparE. dispar cystscysts
stained with trichromestained with trichrome

36. Specific Diagnosis of activeSpecific Diagnosis of active
infection should demonstrateinfection should demonstrate
TrophozoitesTrophozoites
 Motile TrophozoitesMotile Trophozoites
throwing pseudopodiathrowing pseudopodia
and containing red bloodand containing red blood
cells found in largecells found in large
numbernumber
 Endoplasm appearEndoplasm appear
bluish or found glass inbluish or found glass in
appearance and nucleusappearance and nucleus
is not visible but faintis not visible but faint
outline may be observedoutline may be observed

37. Charcot Leyden crystals in stoolCharcot Leyden crystals in stool
examination supports the Diagnosisexamination supports the Diagnosis,,
 Cysts have smooth andCysts have smooth and
thin cell wall and containthin cell wall and contain
round, retractileround, retractile
chromotoid barschromotoid bars
 Glycogen mass is notGlycogen mass is not
visiblevisible
 RBC’s and pus cells areRBC’s and pus cells are
found in fair numberfound in fair number
 Charcot Leyden crystals,Charcot Leyden crystals,
diamond shaped cleardiamond shaped clear
and retractile structuresand retractile structures
are present in faecesare present in faeces
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38. IDOINE PREPARATION OFIDOINE PREPARATION OF
STOOLSTOOL
 Routinely not usedRoutinely not used
 Trophozoites stains yellowTrophozoites stains yellow
to light brown,to light brown,
Nucleus is clearly visibleNucleus is clearly visible
with central karyosomewith central karyosome
Cysts shows a smooth andCysts shows a smooth and
hyaline appearance,hyaline appearance,
Nucleus is clearly seenNucleus is clearly seen
and no more than 4 nucleiand no more than 4 nuclei
are present, Glycogenare present, Glycogen
mass stains brown, whilemass stains brown, while
chromotoid bars are notchromotoid bars are not
stained.stained.

39. Mucosal ScrapingsMucosal Scrapings
 Mucosal scrapings canMucosal scrapings can
be obtained bybe obtained by
sigmoidoscopy useful insigmoidoscopy useful in
atypical presentationsatypical presentations
and may serve as adjunctand may serve as adjunct
to conventional stoolto conventional stool
examination for Ova andexamination for Ova and
cystcyst
 Direct wet mount, aDirect wet mount, a
permanently stainedpermanently stained
smear and immunosmear and immuno
stained smears arestained smears are
examined.examined.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

40. Extra intestinal AmoebiasisExtra intestinal Amoebiasis
 The specimens areThe specimens are
obtained from Liver, lung,obtained from Liver, lung,
or Brain biopsy samplesor Brain biopsy samples
and subjected to routineand subjected to routine
Histopathology ( H&E)Histopathology ( H&E)
sectionssections
 Giemsa stained touchGiemsa stained touch
preparations which willpreparations which will
revel Trophozoites inrevel Trophozoites in
extra intestinal lesions.extra intestinal lesions.

41. Amoebic Liver AbscessAmoebic Liver Abscess
 The pus in liverThe pus in liver
abscess appear asabscess appear as
red Anchovy saucered Anchovy sauce
like appearancelike appearance
 The materialThe material
aspirated is likely toaspirated is likely to
contain Trophozoitescontain Trophozoites
and may be detectedand may be detected
by direct microscopicby direct microscopic
examinationexamination

42. Serological DiagnosisSerological Diagnosis
 The serological become reactive in invasiveThe serological become reactive in invasive
AmoebiasisAmoebiasis
 1 Indirect Heamagglutination assay ( IHA )1 Indirect Heamagglutination assay ( IHA )
 2 ELISA2 ELISA
 3 Latex agglutination test3 Latex agglutination test
 4 gel diffusion4 gel diffusion
 5 Counter current Imunoelectrphoresis5 Counter current Imunoelectrphoresis
 Serological tests remain positive for several years everSerological tests remain positive for several years ever after successfulafter successful
treatmenttreatment

43. CultureCulture
 Cultures are not done routinelyCultures are not done routinely
 Boeck and Drbohlav’s medium modifiedBoeck and Drbohlav’s medium modified
by Laidlaw extensively used for isolationby Laidlaw extensively used for isolation
and maintenance of E.histolytica.and maintenance of E.histolytica.
 Diamonds axenic medium used in studiesDiamonds axenic medium used in studies
on Pathogenicty, antigenicon Pathogenicty, antigenic
characterization and drug sensitivity testscharacterization and drug sensitivity tests
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

44. Do we need culturing forDo we need culturing for
Diagnosis ?Diagnosis ?
 Trying to get theTrying to get the
amoeba to growamoeba to grow
outside the body isoutside the body is
very difficult andvery difficult and
unreliable, and isunreliable, and is
therefore nottherefore not
generally donegenerally done

45. Immunity in AmoebiasisImmunity in Amoebiasis
 Infection with invasiveInfection with invasive
strains of E.histolyticastrains of E.histolytica
induce both Humoralinduce both Humoral
and cellular response.and cellular response.
 Infection offers someInfection offers some
degree of protection.degree of protection.

46. Immunological Tests are notImmunological Tests are not
confirmatory of Acute Infectionsconfirmatory of Acute Infections
 When the body is exposedWhen the body is exposed
to an infection, theto an infection, the
immune system createsimmune system creates
antibodies to fight it off.antibodies to fight it off.
These can be detectedThese can be detected
with a blood test, andwith a blood test, and
provide evidence that theprovide evidence that the
person has been infectedperson has been infected
withwith E. histolyticaE. histolytica ..
Unfortunately, this testUnfortunately, this test
does not distinguishdoes not distinguish
between past and presentbetween past and present
infectioninfection
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

47. Emerging methods inEmerging methods in
DiagnosisDiagnosis
 These are considered theThese are considered the
most useful tests formost useful tests for
detectingdetecting E. histolyticaE. histolytica..
They test directly for theThey test directly for the
parasite itself by exposingparasite itself by exposing
some stool to a strip ofsome stool to a strip of
paper coated withpaper coated with
antibodies. The parasitesantibodies. The parasites
will stick to the antibodieswill stick to the antibodies
on the paper. The teston the paper. The test
distinguishesdistinguishes E. histolyticaE. histolytica
from other parasites.from other parasites.

48. TreatingTreating AmebiasisAmebiasis..
 Frequently, eitherFrequently, either metronidazolemetronidazole (Flagyl) or(Flagyl) or
tinidazoletinidazole (Fasigyn) are used to treat(Fasigyn) are used to treat
Amebiasis. If this does not work,Amebiasis. If this does not work, Chloroquine,Chloroquine,
emetine, and dehydroemetine can be used.emetine, and dehydroemetine can be used.
Eliminating cysts in carriers who do not haveEliminating cysts in carriers who do not have
symptoms is accomplished withsymptoms is accomplished with diloxanidediloxanide
furoate (Furamidefuroate (Furamide ), iodoquinol (Yodoxin), iodoquinol (Yodoxin ),),
andand paromomycin. Nitazoxanideparomomycin. Nitazoxanide is a neweris a newer
drug that shows promise against not onlydrug that shows promise against not only E.E.
histolyticahistolytica but many other parasites as well.but many other parasites as well.

49. Treating extra intestinalTreating extra intestinal
AmoebiasisAmoebiasis
 Amoebic abscess isAmoebic abscess is
treated similarly totreated similarly to
dysentery, withdysentery, with
antibiotics. Sometimesantibiotics. Sometimes
surgical drainage may besurgical drainage may be
performed, but this isperformed, but this is
usually to rule out otherusually to rule out other
(bacterial) causes of(bacterial) causes of
abscess. It is alsoabscess. It is also
performed if an abscessperformed if an abscess
is about to, or has alreadyis about to, or has already
ruptured.ruptured.

50. Preventing AmoebiasisPreventing Amoebiasis
 Drink only bottled or boiled (for 1 minute) water, orDrink only bottled or boiled (for 1 minute) water, or
carbonated (bubbly) drinks in cans or bottles. Fountaincarbonated (bubbly) drinks in cans or bottles. Fountain
drinks and any drinks with ice cubes aredrinks and any drinks with ice cubes are notnot safe. Watersafe. Water
can be made safe by filtering it through an "absolute 1can be made safe by filtering it through an "absolute 1
micron or less" filtermicron or less" filter andand dissolving iodine tablets in thedissolving iodine tablets in the
filtered water.filtered water.
 Avoid fresh fruit or vegetables that were peeled byAvoid fresh fruit or vegetables that were peeled by
someone else.someone else.
 Avoid milk, cheese, or dairy products that may not haveAvoid milk, cheese, or dairy products that may not have
been pasteurized.been pasteurized.
 Avoid anything sold by street vendors.Avoid anything sold by street vendors.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

51. FoodFood safetysafety
 Thoroughly cook all raw foods.Thoroughly cook all raw foods.
 * Thoroughly wash raw* Thoroughly wash raw
vegetables and fruits beforevegetables and fruits before
eating.eating.
 * Reheat food until the internal* Reheat food until the internal
temperature of the foodtemperature of the food
reaches at least 167ºreaches at least 167º
Fahrenheit.Fahrenheit.
 Wash your hands beforeWash your hands before
preparing food, before eating,preparing food, before eating,
after going to the toilet orafter going to the toilet or
changing diapers, afterchanging diapers, after
smoking or after using a tissuesmoking or after using a tissue
or handkerchief.or handkerchief.
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

52. Personal HygienePersonal Hygiene
 Wash hands thoroughlyWash hands thoroughly
with soap and hot runningwith soap and hot running
water for at least 10water for at least 10
seconds after using theseconds after using the
toilet or changing a baby'stoilet or changing a baby's
diaper.diaper.
 Clean bathrooms andClean bathrooms and
toilets often. Pay particulartoilets often. Pay particular
attention to toilet seats andattention to toilet seats and
taps.taps.
 Avoid sharing towels orAvoid sharing towels or
face washers.face washers.

53. VaccinesVaccines
 Vaccines are being developed and testedVaccines are being developed and tested
for the treatment of Amebiasis. Thefor the treatment of Amebiasis. The
vaccine is a modified version of thevaccine is a modified version of the
proteins expressed on the surface of E.proteins expressed on the surface of E.
histolytica. A study in rodents found thathistolytica. A study in rodents found that
the vaccine prevented the formation ofthe vaccine prevented the formation of
liver abscesses, but much more researchliver abscesses, but much more research
is needed to determine if these vaccinesis needed to determine if these vaccines
are useful and safe in humansare useful and safe in humans
 Doctortvrao’s ‘e’ learning seriesDoctortvrao’s ‘e’ learning series

54. Created for AwarenessCreated for Awareness
for Medical andfor Medical and
Paramedical workers inParamedical workers in
Developing WorldDeveloping World
Dr.T.V.Rao MDDr.T.V.Rao MD
EmailEmail
doctortvrao@gmail.comdoctortvrao@gmail.com