Blood,bone marrow,spleen parasites (171)
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Blood,bone marrow,spleen parasites (171) Blood,bone marrow,spleen parasites (171) Document Transcript

  • BLOOD,BONE MARROW, SPLEEN PARASITES
  • BLOOD NEMATODA Order: Filariata
  • BRUGIA MALAYI /WUCHERERIA BANCROFTI
  • Brugia malayi /Wuchereria bancrofti: B.malayi istransmitted by mosquitoes of the genusMansonia, Anopheles and Aedes.W.bancrofti is transmitted by mosquitoes of thegenus Culex, Anopheles and Aedes.Close-up of mosquitoes on human skin.
  • Lymphatic filariases have a wide geographicdistribution.W.bancrofti and B.malayi infect some128 milion people,and about 43 milion havesymptoms.Lymphatic filariases have a widegeographic distribution.B.malayi infection isendemic in Asia(China, Corea, India, Indonesia,Malaysia, Philippines, Sri Lanka).B.timori infectionoccurs in Indonesia (islands of Alor, Flores, Timor).W.bancrofti has a larger distribution : Asia(China, India, Indonesia, Japan, Malaysia,Philippines, South-East Asia,Sri Lanka, TropicalAfrica, Central and South America, Pacific Islands.
  • Brugia malayi: microfilariae measure 270 by 8µm, have a sheath and a tail with terminalconstriction,elongated nuclei and absence ofnuclei in the cephalic space.They have nocturnalperiodicity.(Wet mount preparation).
  • Brugia malayi: The microfilariae are sheathed andcan be distinguished from W.bancrofti for size(275-320x7,5-10), location of nuclei and tailnuclei.(Fresh examination, particular of thecaudal space).
  • Brugia malayi: detail of the cephalic space.Microfilariae are usually nocturnally periodic butsub-periodic strains of B.malayi and W.bancroftiare observed.(wet mount, detail of the cephalicspace of B.malayi microfilaria).
  • Brugia malayi: identification of microfilariae instained smear is possible by observation of thestained sheath (W.bancrofti sheath does notstain).
  • Microfilaria of Wuchereria bancrofti(Giemsa stain, x 400)
  • Brugia malayi: the tail is tapered and present aconstriction.The last two nuclei are divided by theconstriction. The sheath stains pink.(Caudal space of B.malayi, Giemsa stain).
  • Brugia malayi: the cephalic space is longer thanbroad(in W.bancrofti is as long as broad).(Detail of the cephalic space of B.malayi microfilaria,Giemsa stain).
  • Lymphatic filariasis: adults of B.malayi andW.bancrofti live in the lymphatic vessels andlymphnodes where they cause dilatation,inflammatory infiltrates and, at last, blockage of thelymphatic circulation.Adenolymphangitis, orchitis,epididimitis associated with fever are thecommonest manifestation of the acute stage of theinfection;eosinophilia is frequent at this stage.Lymphoedema particularly of the legs and scrotum,hydrocoeles and chyluria are the result of theprogression of the disease;genital manifestationsare frequent in W.bancrofti infections while they arerare during B.malayi infections.-Lymphatic filariasis: elephantiasis of scrotum.-Tanzanian with elephantiasis due to W.bancrofti.-Another Tanzanian patient with elephantiasis dueto W.bancrofti.-Early hydrocoel in a Tanzanian man withW.bancrofti infection
  • Lymphatic filariasis: elephantiasis is the lastconsequence of the swelling of limbs and scrotum.Diethylcarbamazine (DEC), ivermectine andalbendazole used alone or in combination are thedrugs of choice.-Elephantiasis of the limbs.-Thai patient with elephantiasis of leg due toW.bancrofti or Brugia malayi.-Second Thai patient with elephantiasis due tolymphatic filariasis.
  • LOA LOA
  • Loa loa: the infection is endemic in West andCentral Africa,especially in Angola, Cameroun,Congo, Eq. Guinea, Gabon, Nigeria, RCA, Zaire.
  • Loa loa: after injection larvae develop into adultsin 6 months and may live for 17 years in theorganism.Microfilariae measure 275 by 5-6 µmand are present in blood without periodicity.Count is mandatory before therapy.
  • Loa loa: microfilariae measure with the sheath240-300 by 5-6 µm.The sheath doesnt stain withGiemsa. The nuclei extend from the small cephalicspace to the tip of the tail.(Giemsa stain).
  • Loa loa: the sheath does not stain and appearsas a virtual space around the larva.(Giemsa stain).
  • Loa loa: the nuclei form a continuous row to thetip of tail.The unstained sheath is well visible.(Detail of the caudal space, Giemsa stain).
  • Loa loa: large nuclei extend from the littlecephalic space.(Detail of the cephalic space, Giemsa stain).
  • Loa loa: the presence of the unstainded sheath isclearly visible as a space around the larva.(Detail of cephalic space with a polymorphonuclearcell, Giemsa stain).
  • Loa loa: microfilaria.Thick film, Mayers haematoxylin (400 X).
  • Loa loa: microfilaria, tail area.Thick film, Mayers haematoxylin (1.000 X).
  • Loa loa: microfilariae can be demonstrated in bloodwith fluorochromes(Acridine orange stain).
  • Loa loa: although direct diagnosis by observationof microfilariae in blood is the reference method,indirect diagnostic tests such as IF may allowdiagnosis when direct observation is negative,especially in subjects who are not resident inendemic areas.The frequent cross-reaction with other nematodeinfections limits the usefulness of serology inthese patients.Immunodiagnosis by indirectimmunofluorescence.Antigen: frozen sections of Dirofilaria immitis.
  • MANSONELLA OZZARDI MANSONELLA PERSTANSM. ozzardi is endemic in Central and SouthAmerica.The adults live in subcutaneous tissues.Unsheathed microfilariae (200x4-5 µm) released inblood without periodicity,have a small cephalicspace, a long and slender tail without nuclei to theend.
  • M. perstans is endemic in Africa and SouthAmerica. The adult lives in body cavities.Unsheathed microfilariae (200x4-5 µm) releasedin blood without periodicity have a small cephalicspace and nuclei to the end of tail.
  • Mansonella perstans: microfilaria.Thick film, (400 X).
  • BLOOD SPOROZOEAOrder: Eucoccidiida Plasmodium falciparum Plasmodium malariae Plasmodium ovale Plasmodium vivax Toxoplasma gondii
  • PLASMODIUM FALCIPARUM
  • Plasmodium sp.: geographic distribution.
  • Plasmodium sp.: the genus Anopheles includesmore than 400 species of mosquitoes.Manymay act as vectors of human diseases such asmalaria,filariasis and some arbovirus.Eggs present a pair of lateral floats and are laidsingly on the water surface,while larvae lay ina horizontal position under the water surface.
  • Plasmodium sp.: the resting position of the adult ischaracteristic with the proboscid, head andabdomen in a straight line at an angle of about 45°with the surface on which they rest.Only about 60species can transmit malaria and they greatlydiffer in their efficiency as vectors according toman biting behaviour, survival, fertility, adaptationto different breeding place.The most efficientvectors belong to the A.gambiae complex,widelydistributed in tropical Africa, where also importantis A.funestus.In Asia important vectors areA.culicifaciens, A.dirus, A.sinensis and A.miminus;in the Pacific area A.farauti and A.maculatus play apredominant role in malaria transmission. Themain vector in South America in A.albimanus.
  • P.falciparum: species identification is possible onthe basis of the appearance of parasites of each ofthe four malaria species.Shape and size of asexualparasites and of macro- and microgametocytes,developmental stages in peripheral blood,modifications of infected erythrocytes,presence ofdots or clefts on the red blood cells are the maindifferential characteristics.
  • Malaria diagnosis relies on observation ofparasites in Giemsa-stained thin or thick smears(G-TS).Alternative techniques for identification of malariaparasites are based on fluorochromes such asAcridine Orange (AO), DAPI-PI or BCP.With these dyes malaria parasites are easilyrecognized under UV light, reducing the time spentreading the slides.Another method, based onfluorochromes, the quantitative buffy coat (QBC)(Becton-Dickinson) analysis wich uses AO stainingof centrifuged parasites in a capillary tubecontaining a float, has been shown to be rapid andaccurate.
  • P.falciparum: gametocytes of P.falciparum. QBC technique (60X).Recently different immunochromatographictests such as the ParaSight F(Becton Dickinson)and the Malaquick (ICT) wich capture anddetect the histidine rich protein 2 (HRP-2)antigen,and the OPTIMAL wich detectsPlasmodium lactate dehydrogenase(pLDH)have been developed and distributed.The tests are highly sensitive and specific andare now able to distinguish P.falciparuminfections from non-falciparum infections.P.falciparum trophozoites, thin smear, Giemsastain.
  • Malaria diagnosis:whereas thin film gives moreinformations on parasite morphology and permitsan easier morphologic differentiation,G-TS ismore sensitive allowing a concentration ofplasmodia (10-15 folds) and it is the standardreference diagnostic test.
  • P.falciparum trophozoites, thick smear, Giemsa stain.Malaria diagnosis:G-TS needs careful stain (2% Giemsa) and experience inexamining slides;reasonable sensitivity is reached byobserving at least 500-1.000 White Blood Cells(WBC).Quantification of baseline parasitemia is necessaryfor monitoring the response to therapy. Parasites must becounted in parallel with leucocyte and parasitemiaexpressed as parasites/µl. N. of parasites counted x N. of WBC/µl = N. of parasites/µl N. of WBC counted
  • P. falciparum: trophozoites are small rings withsingle or double small chromatin dots, andregular cytoplasm; multiple infection andhigh parasitemia (>5%) are common.Dots or cleft (Maurers) can be observed on theinfected RBCs.P.falciparum trophozoites, thin smear, Giemsastain.
  • P. falciparum: sometimes trophozoites appear atthe edge of the red blood cell (applique form)left.Erythrocytes maintain regular shape andsize.P.falciparum trophozoites, thin smear, Giemsastain.
  • P. falciparum: late trophozoites and schizontsusually are not observed in peripheral bloodunless in severe infections.Cerebral malaria: late trophozoites with acoarse granule of pigment in peripheral blood.P.falciparum, thin smear, Giemsa stain.
  • P.falciparum: micro- and macrogametocytes areeasily recognized by their crescentic, cigar- orbanana-like shape.Microgametocytes have adiffuse chromatin,while macrogametocytes havethickened chromatin.Microgametocyte, Giemsa thin smear.
  • P.falciparum: in thick films red blood cells are notvisible and leucocytes and parasites appearsmaller than in thin smears.Trophozoites have aring or comma shape, with one or two dots ofchromatin.The pigment, when present, is compact.
  • P.falciparum: trophozoites in Giemsa-stainedthick films have a wide range of shapes.Maurers clefts are not visible.
  • P.falciparum: micro- and macrogametocytes havean evident malaric pigment,scattered through inthe cytoplasm in the microgametocyte.Microgametocyte, Giemsa thick smear.
  • P.falciparum: staining with fluorochromes is rapid(less than 1 min)and observation of slides can beperformed at low magnification (400X) allowingrapid screening of smears even with lowparasitemia. P.falciparum (DAPI-PI).
  • P.falciparum trophozoites. Acridine Orange stain.
  • P.falciparum: the sensitivity of different isolatesof P.falciparum to drugs can be assessed with theWHO "in vitro test".The development to mature schizont in presenceof therapeutic levels of the drug demonstratesresistance of the isolate.
  • P.falciparum: severe P.falciparum infections areclinical forms characterized by potentially fatalmanifestations or complications:cerebral malaria,defined by a state of unrousable coma in absenceof other causes,is the most common manifestation.Celebral malaria: parasitized RBCs in brain vessels(H&E stain).
  • P.falciparum: rosetting of infected and uninfectedred blood cells and cytoadherence of parasitizederythrocytes to the vascular endothelium,play acrucial role in sequestration of parasites andobstruction of brain vessels.Induction of host cytokines and soluble mediatorssuch as oxygen radicals and NO play an importantrole in the pathogenesis of the infection.
  • P.falciparum: the brain appears oedematous,hyperaemic and with pigment deposition; thecapillaries, expecially of the white matter,appear dilated and congested and obstructed byparasitized RBCs.
  • P.falciparum: renal failure may result fromsequestration of RBCs and alteration of the renalmicrocirculation.Glomerulal and interstitial vessels present RBCsadhering to the endothelium.
  • P.falciparum: renal failure may also result fromreleasing of compounds secondary to intravascularhaemolysis (not haemoglobin itself) that can causeacute tubular necrosis especially in presence ofdehydratation and acidosis.
  • P.falciparum: sequestration and cytoadherenceof parasitized RBCs in heart microcirculation isfrequent but myocardial dysfunctions andcardiac arrhythmias are uncommon in severefalciparum malaria.
  • P.falciparum: jaundice and abnormalities of liverfunction tests are frequent findings in severefalciparum malaria but hepatic failure is rare evenin heavily infected individuals.
  • P.falciparum: histological abnormalities includeKuppfer hyperplasia,mononuclear hyperplasia andsinusoid dilatation;swollen hepatocytes containhaemosiderin.Kuppfer cells contain a lot ofmalaria pigment.
  • A fatal case of P.falciparum malaria (liver):malarial pigment within Kupffer cells (H&E X 400)
  • A fatal case of P.falciparum malaria (liver):note a parasitized erytrocyte (H&E X1000)
  • P.falciparum: pulmonary, non specific complications,such as atypical pneumonia, lobar pneumonia orbronchopneumonia,frequently occur during malariainfections.Pulmonary oedema is a specific and severecomplication of P.falciparum infection: 3-10% Thissyndrome, wich resembles the Acute RespiratoryDistress Syndrome (ARDS),has a relative late onset(wich may be abrupt) in the course of the infection andis often associated with other manifestations of thesevere falciparum malaria.Different pathogenicmechanisms have been suggested:-increased capillary membrane permeability[due tomicroemboli or to Disseminated IntravascularCoagulation (DIC)]-impaired function of the alveolar capillaries;-severe disfunction of the pulmonary microcirculation;-allergic phenomena;-therapeutic fluid overload.The chest radiograph, in severe cases, showswidespread bilateral,confluent intraalveolar andinterstitial infiltrates.
  • PLASMODIUM MALARIAE
  • Plasmodium sp.: geographic distribution.
  • Plasmodium sp.: the genus Anopheles includesmore than 400 species of mosquitoes.Many mayact as vectors of human diseases such as malaria,filariasis and some arbovirus.Eggs present a pair of lateral floats and are laidsingly on the water surface,while larvae lay in ahorizontal position under the water surface.
  • Plasmodium sp.: the resting position of the adult ischaracteristic with the proboscid, head and abdomenin a straight line at an angle of about 45° with thesurface on which they rest.Only about 60 species cantransmit malaria and they greatly differ in theirefficiency as vectors according to man bitingbehaviour, survival, fertility, adaptation to differentbreeding place.The most efficient vectors belong tothe A.gambiae complex,widely distributed in tropicalAfrica, where also important is A.funestus.In Asia important vectors are A.culicifaciens, A.dirus,A.sinensis and A.miminus;in the Pacific areaA.farauti and A.maculatus play a predominant rolein malaria transmission. The main vector in SouthAmerica in A.albimanus.
  • P.malariae: species identification is possible on thebasis of the appearance of parasites of each of thefour malaria species.Shape and size of asexualparasites and of macro- and microgametocytes,developmental stages in peripheral blood,modifications of infected erythrocytes,presence ofdots or clefts on the red blood cells are the maindifferential characteristics.
  • Malaria diagnosis relies mainly on observation ofparasites in Giemsa-stained thin or thick smears(G-TS).Alternative techniques for identification ofmalaria parasites are based on fluorochromessuch as Acridine Orange (AO), DAPI-PI or BCP.With these dyes malaria parasites are easilyrecognized under UV light,reducing the time spentreading the slides.Another method, based onfluorochromes, the quantitative buffy coat (QBC)(Becton-Dickinson) analysis wich uses AO stainingof centrifuged parasites in a capillary tubecontaining a float, has been shown to be rapid andaccurate.
  • P.falciparum: gametocytes of P.falciparum. QBC technique (60X).Recently different immunochromatographic testssuch as the ParaSight F(Becton Dickinson) and theMalaquick (ICT) wich capture and detect thehistidine rich protein 2 (HRP-2) antigen, and theOPTIMAL wich detects Plasmodium lactatedehydrogenase (pLDH) have been developed anddistributed.The tests are highly sensitive and specific and arenow able to distinguish P.falciparum infectionsfrom non-falciparum infections.P.malariae trophozoites, thin smear, Giemsa stain.
  • Malaria diagnosis:whereas thin film gives more informations onparasite morphology and permits an easiermorphologic differentiation, G-TS is more sensitiveallowing a concentration of plasmodia (10-15folds)and it is the standard reference diagnostic test.
  • trophozoites, thick smearSchizont,thick smear Malaria diagnosis: G-TS needs careful stain (2% Giemsa) and experience in examining slides; reasonable sensitivity is reached by observing at least 500-1.000 White Blood Cells (WBC). Quantification of baseline parasitemia is necessary for monitoring the response to therapy. Parasites must be counted in parallel with leucocyte and parasitemia expressed as parasites/µl. N. of parasites counted x N. of WBC/µl = N. of parasites/µl N. of WBC counted
  • P. malariae: trophozoites are usually small ringswith a single dot of chromatin or have a compact,regular cytoplasm that seems to contain thenucleus.The pigment in late trophozoites is scattered.(Thin smear, Giemsa).
  • P. malariae: trophozoites may assume a band formtypical of the species.Red blood cells are notenlarged or rather smaller than normal.Multipleinfection is rare. The parasitemia is usually low. Nodots or clefts.(Thin smear, Giemsa).
  • P.malariae: schizonts are small and with a lownumber of merozoites (<12) arranged in regularforms (rosettes) with a thickened, often central,pigment.The complete erythrocytic cycle takes72 hours and ends with the releasing of freemerozoites (c).(Thin smear, Giemsa).
  • P. malariae: micro- and macrogametocytes areround, small with chromatin defined; they mustbe differentiated from late trophozoites.DuringP.malariae infection all stages of developmentare present in peripheral blood.(Microgametocyte, Giemsa stain).
  • PLASMODIUM OVALE
  • Plasmodium sp.: geographic distribution.
  • Plasmodium sp.: the genus Anopheles includesmore than 400 species of mosquitoes.Many mayact as vectors of human diseases such as malaria,filariasis and some arbovirus.Eggs present a pair of lateral floats and are laidsingly on the water surface,while larvae lay in ahorizontal position under the water surface.
  • Plasmodium sp.: the resting position of the adult ischaracteristic with the proboscid, head and abdomenin a straight line at an angle of about 45° with thesurface on which they rest.Only about 60 species cantransmit malaria and they greatly differ in theirefficiency as vectors according to man bitingbehaviour, survival, fertility, adaptation to differentbreeding place.The most efficient vectors belong tothe A.gambiae complex,widely distributed in tropicalAfrica, where also important is A.funestus.In Asia important vectors are A.culicifaciens, A.dirus,A.sinensis and A.miminus;in the Pacific areaA.farauti and A.maculatus play a predominant rolein malaria transmission. The main vector in SouthAmerica in A.albimanus.
  • P.ovale: species identification is possible on thebasis of the appearance of parasites of each of thefour malaria species.Shape and size of asexualparasites and of macro- and microgametocytes,developmental stages in peripheral blood,modifications of infected erythrocytes,presence ofdots or clefts on the red blood cells are the maindifferential characteristics.
  • Malaria diagnosis relies on observation ofparasites in Giemsa-stained thin or thick smears(G-TS).Alternative techniques for identification ofmalaria parasites are based on fluorochromessuch as Acridine Orange (AO), DAPI-PI or BCP.With these dyes malaria parasites are easilyrecognized under UV light,reducing the timespent reading the slides.Another method, basedon fluorochromes, the quantitative buffy coat(QBC)(Becton-Dickinson) analysis wich uses AOstaining of centrifuged parasites in a capillarytube containing a float, has been shown to berapid and accurate.
  • Recently different immunochromatographic testssuch as the ParaSight F (Becton Dickinson) andthe Malaquick (ICT) wich capture and detectthe histidine rich protein 2 (HRP-2) antigen, andthe OPTIMAL wich detects Plasmodium lactatedehydrogenase (pLDH) have been developed anddistributed.The tests are highly sensitive andspecific and are now able to distinguishP.falciparum infections from non-falciparuminfections.P.ovale, thin smear, Giemsa stain.
  • Malaria diagnosis:whereas thin film gives more informations onparasite morphology and permits an easiermorphologic differentiation, G-TS is moresensitive allowing a concentration of plasmodia(10-15 folds)and it is the standard referencediagnostic test.
  • P.ovale, thick smear, Giemsa stain.Malaria diagnosis: G-TS needs careful stain(2% Giemsa) and experience in examining slides;reasonable sensitivity is reached by observing at least500-1.000 White Blood Cells (WBC).Quantification of baseline parasitemia is necessaryfor monitoring the response to therapy.Parasites must be counted in parallel with leucocyteand parasitemia expressed as parasites/µl. N. of parasites counted x N. of WBC/µl = N. of parasites/µl N. of WBC counted
  • Plasmodium ovale: trophozoiteAll stages are seen in blood films; prominentShuffners dots are present at all stages.Trophozoites appear as rings with, usually, acompact cytoplasm;they do not have ameboidcytoplasm.The parasites are smaller than P.vivax.
  • P.ovale: red blood cells are enlarged, ovalizedand distorted with fimbriae at poles.Schizonts have usually 8-10 merozoites.
  • P.ovale: micro- and macrogametocytes aresometimes difficult to differentiate from latetrophozoites;they are round and occupy almostthe entire erythrocyte.Microgametocytes have amore scattered chromatin.
  • PLASMODIUM VIVAX
  • Plasmodium sp.: geographic distribution.
  • Plasmodium sp.: the genus Anopheles includesmore than 400 species of mosquitoes.Many mayact as vectors of human diseases such as malaria,filariasis and some arbovirus.Eggs present a pair of lateral floats and are laidsingly on the water surface,while larvae lay in ahorizontal position under the water surface.
  • Plasmodium sp.: the resting position of the adult ischaracteristic with the proboscid, head and abdomenin a straight line at an angle of about 45° with thesurface on which they rest.Only about 60 species cantransmit malaria and they greatly differ in theirefficiency as vectors according to man bitingbehaviour, survival, fertility, adaptation to differentbreeding place.The most efficient vectors belong tothe A.gambiae complex,widely distributed in tropicalAfrica, where also important is A.funestus.In Asia important vectors are A.culicifaciens, A.dirus,A.sinensis and A.miminus;in the Pacific areaA.farauti and A.maculatus play a predominant rolein malaria transmission. The main vector in SouthAmerica in A.albimanus.
  • P.vivax: species identification is possible on thebasis of the appearance of parasites of each ofthe four malaria species. Shape and size ofasexual parasites and of macro- andmicrogametocytes,developmental stages in peripheral blood,modifications of infected erythrocytes,presenceof dots or clefts on the red blood cells are themain differential characteristics.
  • Malaria diagnosis relies mainly on observation ofparasites in Giemsa-stained thin or thick smears(G-TS).Alternative techniques for identification ofmalaria parasites are based on fluorochromessuch as Acridine Orange (AO), DAPI-PI or BCP.With these dyes malaria parasites are easilyrecognized under UV light, reducing the timespent reading the slides.Another method, basedon fluorochromes, the quantitative buffy coat(QBC) (Becton-Dickinson) analysis wich uses AOstaining of centrifuged parasites in a capillarytube containing a float,has been shown to berapid and accurate.
  • Recently different immunochromatographic testssuch as the ParaSight F(Becton Dickinson) andthe Malaquick (ICT) wich capture and detectthe histidine rich protein 2 (HRP-2) antigen,and the OPTIMAL wich detects Plasmodiumlactate dehydrogenase (pLDH)have beendeveloped and distributed.The tests are highlysensitive and specific and are now able todistinguish P.falciparum infections from non-falciparum infections.P.vivax trophozoites, GT-s.
  • Malaria diagnosis:whereas thin film gives more informations onparasite morphology and permits an easiermorphologic differentiation,G-TS is more sensitiveallowing a concentration of plasmodia(10-15 folds)and it is the standard reference diagnostic test.
  • P. vivax trophozoites, thick smear, Giemsa stain.Malaria diagnosis:G-TS needs careful stain (2% Giemsa) and experience inexamining slides;reasonable sensitivity is reached byobserving at least 500-1.000 White Blood Cells (WBC).Quantification of baseline parasitemia is necessaryfor monitoring the response to therapy.Parasites must becounted in parallel with leucocyte and parasitemiaexpressed as parasites/µl. N. of parasites counted x N. of WBC/µl = N. of parasites/µl N. of WBC counted
  • P.vivax: young trophozoites are small with single(rarely double) chromatin,with a loop of thincytoplasm.The red blood cell is sligthly enlargedand a few Shuffners dots are present.Parasitemia range form 0.5 to 2%, multipleinfection is rare.
  • P.vivax: the trophozoites increases in size and thecytoplasm becomes ameboid with rapid movements("vivax").The red blood cell enlarges and prominetShuffners dots are present.(Thin smear, Giemsa).
  • P.vivax: in more advanced stage of developmenttrophozoites occupy most of the RBC, and have alarge vacuole and fine rods of pigment.Thenucleus increases in size.
  • P.vivax: late trophozoites have a more densecytoplasm, and a large vacuole.
  • P.vivax: in young schizonts the nucleus divides andthe vacuole disappears;the cytoplasm is dense.
  • P.vivax: in about 48hours schizogony iscompleted.Mature schizont may contain 12-24merozoites. In thick smears schizonts looksmaller than in thin smears and the Schuffnersdots are not always visible.
  • P.vivax: gametocytes are round or oval withoutvacuole; most of the RBC is occupied by theparasite. Macrogametocytes have a compactchromatin mass while microgametocytes have amore diffuse nucleus stained pink.
  • P.vivax: staining with fluorochromes is rapid (lessthan 1 min) and observation of slides can beperformed at low magnification (400X) allowing rapidscreening of smears even with low parasitemia.P. vivax (DAPI-PI).
  • TOXOPLASMA GONDII
  • T. gondii: T.gondii encephalitis (TE) is the mostcommon cerebral opportunistic infection inpatients with AIDS.The typical lesion is an ipodense focal area withring contrast-enhancement and edema.(CT scan of a toxoplasmic encephalitis).
  • T. gondii: tissue cysts, 100-300 µm, may containup to 3.000 bradyzoites.The wall of maturepseudocysts is believed to represent acombination of host and parasitic components.
  • T. gondii: diagnosis of TE is usually presumptive,based on clinical and radiologic findings and on theresponse to treatment; cerebral biopsy sometimesallows identification of pseudocysts in tissuesections. (H&E stain).
  • T. gondii: toxoplasmic pseudocyst within aninflammatory tissue reaction. (H&E stain).
  • T. gondii: the pseudocysts of T.gondii can beobserved in tissue sections with monoclonalantibodies.
  • T. gondii: direct detection of T.gondii in clinicalspecimens is rare;parasites can be isolated fromblood, CSF, amniotic fluid,tissue biopsies on cell lines(THP-1 or MRC-5).In clinical specimens the presence of parasites canalso be demonstrated by PCR analysis.
  • T. gondii: intracellular trophozoites of T.gondii in acell culture.The trophozoites proliferate within the vacuoledeveloping a pseudocyst.(Trophozoites in a THP-1 cell, Giemsa stain).
  • T. gondii: in cell cultures T.gondii proliferates toform a pseudocyst of 8-20 parasites.(Trophozoites in a THP-1 cell, Giemsa stain).
  • T. gondii: lysis of a THP-1 cell with release oftachizoites in culture.(Trophozoites in a THP-1 cell, Giemsa stain).
  • T. gondii: microscopical features of tachizoites ofToxoplasma gondii and peritoneal macrophagesof mouse in peritoneal exudate. (SEM)
  • T. gondii: microscopical features of tachizoites ofToxoplasma gondii and peritoneal macrophages ofmouse in peritoneal exudate. (SEM)
  • T. gondii: the anterior pole of an endozoid intangential projection.Several subpellicularfibrils and their insertion on the anterior polarring are visible.
  • T. gondii: transmision electron microscopic picture.Longitudinal section of an endozoid.
  • T. gondii: cross-section through an endozoidin an advanced stage of endodiogeny.The daugther cells appear to be surrounded.In each of these news cells there are two roundbodies that lengthen forming the first rhoptries.
  • SPOROZOEAOrder :Piroplasmida
  • BABESIA CANISBabesia spp: babesiosis is a zoonosis that affectsseveral animals:B.canis (dogs), B.equi (horses),B.bovis (cattle), B.microti (rodents).Some Babesiaspp. are not host specific and can be transmitted tohumans:B. microti and B.bovis/divergens.Theinfection is transmitted by the bite of ticks of theFamily Ixodidae of the genera Dermatocentor,Ixodes and Rhipicephalus.The main vector ofB.microti is I.dammini, while vector of B.microti isI.ricinusB.canis, Giemsa stain.
  • Babesia spp.: intraerythrocytic organisms in bloodsmears
  • Babesia spp.: after inoculation by the vector, thetrophozoites enter the bloodstream and multiply inside theerythrocytes by budding, releasing two to fours daughterparasites and causing hemolytic anemia. Ticks becomeinfected by ingesting blood of parasitized mammals.Motile "vermicules" develop and multiply in the ticks gutand then migrate through the body (salivary glands andovaries).In some species transovarial transmission(B.bovis and B.caballi)or transtadial passage, from larva tonimph (B.microti) occur.Vermicules of Babesia spp.(B.caballi ?) obtained from crushed Rhipicephalusturanicus eggs. Tick collected from horses in a militaryfarm in Turkey where the prevalence of equine babesiosisis high.
  • Babesia spp.: by transovarial transmission "vermicules"can infect tick eggs;they multiply in the yolk and in intestinal tissues of thelarva;pyriform bodies are then observed in the salivary glands ofthe haematophage larvae and nimphs.Vermicules of Babesia spp. (B.caballi ?) obtained fromcrushed Rhipicephalus turanicus eggs. Tick collected fromhorsesin a military farm in Turkey where the prevalence ofequine babesiosis is high.
  • B.canis: diagnosis depends on the observationof the intraerythrocytic organisms in bloodsmears.Pear shaped microorganisms (2-5 µm)and tetrads are the diagnostic shape of theparasite. (Giemsa stain).
  • B.canis: intraerythrocytic parasites can beconfused with P.falciparum or P.malariaetrophozoites.Ring and band forms are sometimes observed.(Giemsa stain).
  • B.equi: trophozoites of B.equi can mimicP.falciparum young ring trophozoites.
  • ZOOMASTIGOPHOREA Order: Kinetoplastida
  • TRYPANOSOMA CRUZI (Chagas disease)T. cruzi: american trypanosomiasis was firstdescribed by Carlos Chagas in Brasil in 1909.The infection, Chagas disease, is causedby the haemoflagellate Trypanosoma cruzi.tc1: T.cruzi in blood sample, Giemsa.
  • T. cruzi: the disease is a public health threat inmost Latin American countries,although cases dueto blood derivatives or blood transfusion has beenreported in non-endemic regions.According to WHO the overall prevalence of humanT.cruzi infection is estimated in 18 million casesand 100 million people are living at risk.tc2: T. cruzi: geographical distribution.
  • T. cruzi: the vectors are reduvidae bugs which arehaematophagus and the most important areTriatoma infestans(Argentina, Chile, Brazil, Bolivia,Paraguay, Uruguay, Peru),T. sordida (Argentina,Bolivia, Brazil, Paraguay),Rhodnius prolixus(Colombia, Venezuela, Mexico, Central America),T. dimidiata (Ecuador, Mexico, Central America),and Panstrogylus megistus (northeast Brazil).
  • T. cruzi: the transmission by the vector is faecal.T.cruzi infective metacyclic trypomastigotes are shedin the faeces of the bug and are inoculated into thehuman host by scratching infected faeces into skinabrasions usually caused by the bug in the process offeeding (blood-sucking). T.cruzi metacyclic trypomastigote: scanning electronmicroscopy showing T.cruzi trypomastigotesrecovered from an infected Triatoma spp. in PedroCarbo, Ecuador.
  • T. cruzi: infective metacyclic trypomastigotes areshed in the faeces of the bug and inoculated intothe vertebrate host not only by skin lesions but alsothrough the mucosa of the mouth and,in humans,through the conjunctiva of the eyes.
  • T. cruzi: trypomastigotes can infect most of thevertebrate cells,polymorphonuclear leucocytes andmacrophages are probably among the firstvertebrate host cells with which T.cruzi interacts invivo.tc7a: In vitro T.cruzi infection of macrophagesshowing the presence of amastigotes:Wright-Giemsa stain, showing replicating T.cruziamastigotes within host cell.
  • T. cruzi: this invasive step is crucial for the lifecycle of the parasite since it has to becomeintracellular to multiply.tc7b: In vitro T.cruzi infection of macrophagesshowing the presence of amastigotes:immunofluorescence assay showing T.cruziamastigotes after treatment with anti-T.cruzipolyclonal mouse sera.
  • T. cruzi: trypomastigotes in the host cell transforminto amastigotes,which multiply intracellularly bybinary division inducing inflammatory andimmunological responses in vivo, and destroy cellsin vitro.Amastigotes are then released into the bloodstream as trypomastigotes.The latter arenondividing forms which are able to infect a widerange of new host cells but muscle and glia seemmost often parasitized,or they have to be ingestedby another reduviid bug in order to continue theparasite life cycle in the invertebrate host.tc8: Trypomastigotes reach the myocardial cellsand after penetration they multiply as amastigoteswith formation of a pseudocyst.
  • T. cruzi: in the Reduvidae bug the bloodstreamderived trypomastigote forms pass along thedigestive tract through irreversible morphologicaltransformations in sequence;each developmentalstage occurs in a specific portion of the insects gut.Thus, in the stomach, most blood trypomastigoteschange into epimastigotes and rounded forms(sphaeromastigotes).tc9: T.cruzi epimastigote. Immunofluorescencestudies using antibodies to a T.cruzi protein namedTc52(immunosuppressive factor which also express athiol-transferase activity)and confocal microscopy.An intense labeling located at the posterior end ofan epimastigote indicate that Tc52 is targeted to thereservosomes(These organelles are small vesiclesinside multivesicular structures being formedpredominantly at the posterior end of epimastigotes).
  • T. cruzi: epimastigotes divide actively in thevectors intestine and reach the rectum wherea final differentiation results in the infectivemetacyclic trypomastigotes which areeliminated in the bugs faeces.tc10: T.cruzi epimastigote. Epimastigotereacting with a monoclonal antibody againstT.cruzi.
  • T. cruzi: some researchers have postulated thatsphaeromastigotes may change either into shortepimastigotes,dividing forms in the intestine, orinto long epimastigotes which are nondividingforms but are able to reach the rectum where theytransform into the final metacyclic trypomastigoteform.In any case, this hypothesis remainscontroversial.tc10b: T.cruzi epimastigote. Scanning electronmicroscopyshowing T.cruzi epimastigote.
  • T. cruzi: there are three phases of the infection.The acute phase usually passes unnoticed butthere may be an inflamed swelling or chagomaat the site of entry of the trypanosomes.Romanassign is when this swelling involves theeyelids but it occurs only in about 1-2% of thecases.In the acute phase, mortality is less than 5%and death may result from acute heart failureor meningoencephalitis in children less than twoyears old.Romana’s sign, clinical manifestationtipically observed in the acute phase of someChagas’ disease patients.
  • T. cruzi: general symptoms in acute Chagas diseasemay also include fever, hepatosplenomegaly,adenopathies and myocarditis.Electrocardiographicchanges involve sinus tachycardia, prolongationof the P-R interval, primary T-wave changes andlow QRS voltage.Chest X-ray can revealcardiomegaly of different degrees.The intermediate phase is clinically asymptomaticand is detected by the presence of specificantibodies.No parasites are found in bloostreamsmears but xenodiagnosis could be positive in somecases.Acute Chagas myocarditis (Haematoxylin and EosinX 160)tc12: Posteroanterior chest radiographshowing enlarged heart due to T.cruzi infection.tc12a: Acute Chagas disease myocarditis(Haematoxylin and Eosin X160)
  • T. cruzi: the chronic phase of Chagasdiseasedevelops 10 - 20 years after infection and affectsinternal organs such as the heart,oesophagus andcolon as well as the peripheral nervous system.The lesions of Chagas’ disease are incurable and insevere cases patients may die as result of heartfailure. T.cruzi parasitize mainly the cardiac muscle but any cell type may be parasitized (smooth muscle cells, hystiocytes): cardiac muscle with amastigotes, H&E stain.
  • Chagas disease megacardia(slide from the late Prof.Koberle, Brazil)
  • Apical aneurysm in Chagas disease(slide from the late Prof.Koberle, Brazil)
  • T. cruzi: on the other side, megacolon is associatedwith abnormal constipation (weeks).Faecalimpaction and sigmoid volvulus are side-effects ofmegacolon.Neurological changes in chronicChagas disease include changes at the level of thecentral, peripheral or autonomic nervous system. Chagasic megacolon with enlargement of the sigmoid;patient from Morona Santiago province, southeastern Ecuador
  • X-ray showing megaoesophagus in Chagas disease
  • X-ray showing megacolon in Chagas disease
  • T. cruzi: can be observed in the peripheral bloodonly in the acute case of the disease.Its presence isthe best definition of the acute phase as all othersigns are variable.-Wright-Giemsa staining of T.cruzi trypomastigotein peripheral blood smear from an acute infectedpatient.-T.cruzi in mouse blood (Giemsa stain)
  • T. cruzi: trypomastigotes have a prominentsubterminal kinetoplast that often distort themembrane of the cell,an elongated nucleus andan undulating membrane.-T.cruzi trypomastigote: blood streamtrypomastigotes are 15-20 µm in length andappear with a typical C or S-shaped form.
  • T. cruzi: multiplication only occurs in theamastigote phase,which grows in a variety of tissue cells especiallymuscle.-In vitro infected fibroblast showing a largenumber of intracellular amastigotes.
  • T. cruzi: laboratory diagnostic tests based onserology (IFA, ELISA) and Polymerase ChainReaction (PCR) specific for T.cruzi, have beendeveloped.-T.cruzi trypomastigotes reacting with monoclonalAb.
  • T. cruzi: serological cross-reactions can occurwith infections such as leprosy, leishmaniasis,treponematoses, malaria and multiple myeloma.Trypanosoma rangeli is also an important causeof false-positive testing, especially in areas whereT.cruzi coexists with T.rangeli.-In vitro T.cruzi infection of macrophagesshowing the presence of amastigotes:confocal microscopy showing T.cruzi amastigotesafter treatment with anti-Tc24 mouse sera.
  • T. cruzi: two drugs are in common use.Nifurtimox (Lampit, production was discontinued in1991)and Benznidazole (Rochagan).The latter which is now the drug of choice,is given in an oral dose of 6 mg/kg body weight for30 or 60 days.Both drugs produce anorexia, weightloss, headache and dizziness,gastric irritation, andsometimes peripheral neuritis.Experimental drugsare under evaluation.Treatment of patients in theintermediate or chronic phase is controversial.Congenital Chagasdisease and transfusion-associated acute disease require Rochagantherapy.Transfusion infection can be prevented bydonor screening or,by mixing the blood withgentian violet (0,25 gr./L for 24 hours) to killT.cruzi.Vector control programmes involvinginsecticide spraying with modern pyretroids andnew tools for delivery in endemic areas is beingcarried out in some Latin American countries.tc20: TEM microphotograph of T.cruziepimastigote.
  • ZOOMASTIGOPHOREAOrder: Kinetoplastida
  • TRYPANOSOMABRUCEI RHODESIENSE / T.B. GAMBIENSE
  • Sleeping sickness occurs in Africa between the15° North and the 20° South.The T.b.rhodesiense form is found in East andCentral-East Africa whereas the T.b.gambienseinfection occurs in Central and West Africa.
  • The Africantrypanosomiasisis transmittedby severalspecies of tse-tse flies(Glossina spp.). Larva and pupae of Glossina morsitans Adult Glossina tachinoides in West Africa
  • T. b. gambiense and rhodesiense: two forms oftrypomastigote can be seen in peripheral blood:one is long slender, 30 µm in length,and iscapable of multiplying in the host, the other isstumpy, not dividing,18 µm in length.
  • Trypanosoma brucei gambiense and rhodesiense:trypanosomes appear in the peripheral blood 5 to21 days after the infecting bite.
  • Trypanosoma brucei gambiense and rhodesiense:the terminal stage of the infection ("sleepingsickness") is the result of a chronicmeningoencephalomyelitis. (H&E stain).
  • Trypanosoma brucei gambiense andrhodesiense: the typical pathological lesion oftrypanosomiasis is a perivascular round-cellinfiltration (perivascular cuffing) due to glialcells, lymphocytes and plasmocytes (Mott cells).(H&E stain).
  • LEISHMANIA DONOVANI
  • Visceral leishmaniasis has a wide geographicdistribution.North-Eastern China, India, Middle-East, Southern Europe (Mediterranean bassin),Northern Africa, Central-East Africa and, in foci,Central and South America(especially Brazil andHonduras).
  • The infection is transmitted by various species ofPhlebotomus, the sand fly.
  • Leishamnia spp. wich affect humans can bedifferentiated by geographical distribution, clinicalspectrum, immunological features,isoenzymes andKinetoplast DNA (kDNA) characterization.(Leishmania amastigotes, bone marrow aspirate,Giemsa stain).
  • Visceral leishmaniasis (Kala-azar) is caused byparasites of the genus Leishmania, subgenusLeishmania, complex donovani (donovani,infantum, chagasi species).Viscerotropic strains ofL.infantum and L.tropica have been described.(bone marrow aspirate)
  • Diagnosis of the infection depends onidentification of amastigotes in tissues (bonemarrow, spleen, liver, limph nodes) or inblood.Other organs may be affected, expecially inHIV-1 positive patients(intestinal and respiratory tract).Amastigotes canbe found inside and outside the reticuloendothelialcells. They measure 2-5 µm, are oval with a largenucleus (in red), a kinetoplast (usuallyperpendicular, in red to violet) and a pale bluecytoplasm.(Bone marrow aspirate).
  • Leishamnia sp.: Cultures (on NNN or Tobie media) ofblood or tissues samples may permit isolation of theparasite, allowing the subsequent characterisation.When introduced in culture the amastigotestransform into promastigotes in 7-21 days.(Wet mount preparation).
  • Leishamnia sp.: Leishmania promastigotes measure15-20 by 1.3-3.5 µm and have a single flagellum,measuring 15-28 µm.Serologic examination (EIA,direct Agglutination, IF, WB) is useful inimmunocompetent individuals, not ALWAYS inHIV-1 positive patients.
  • Visceral leishmaniasis: liver biopsy candemonstrate the Leishmania amastigotesinside the reticuloendothelial cells. The hepaticstructure is preserved.
  • Visceral leishmaniasis: liver biopsy at highermagnification with intracellular amastigotes.
  • Visceral leishmaniasis: spleen biopsy is a veryhigh sensitive method of diagnosis but it is notwidely used because of the risk of hemorrhage.Splenic tissue is rich in amastigotes allowing arapid and sensitive diagnosis.
  • Visceral leishmaniasis: spleen biopsy withintracellular amastigotes.