Infection Case ► Int 1 曾耀弘 Instructed by : Professor 盧章智 Date:2005/12/12

Case ► A 35 year old male medical student from Kenya, who was studying in the United States, presented to a hospital ER room in Boston, Mass, after a trip home to visit relatives. ► His symptoms consisted of nausea, vomiting, fever, malaise, night sweats, and severe shaking chills.

► These symptoms had persisted for several weeks, chills and fever occurred periodically, usually at 36 to 48h intervals. ► An enlarged spleen was detected on PE examination. ► Blood was drawn for lab tests, including platelet count, a complete blood count and thick and thin blood smears.

► The results of the CBC revealed that the patient was anemic (hemoglobin, 9.0g/dl) and thrombocytopenic ( platelets, 30,000/ul). ► Thick and thin smears were prepared and stained using the Giemsa method.

► Microscopic examination of these films revealed the presence of a large number (average of three parasites per oil immersion field of intra erythrocytic parasitic forms, mostly in the shapes of small rings.

► A few cells contained multiple rings. A rare banana-shaped gametocyte was seen some early ring trophozoites were identified as applique forms. ► Several blue rings with two red chromatin dots were seen.

► A small number of irregular dark bluish red dots were also seen in the erythrocytes. ► The red blood cells were normal in size and exhibited no stippling. ► Based on these findings a diagnosis of infection with a blood parasite was made

questions ► 1. which infection does this patient have ? ► Ans: Malaria ► Which parasite is infecting him? ► Ans: plasmodia falciparum.

► presence of dots or clefts on the red blood cells are the main differential characteristics. ► A rare banana-shaped gametocyte was seen some early ring trophozoites were identified as applique forms.

Describe the typical appearance of this parasite in thick and thin Giemsa stained smears. What are the red dots seen in the smear? ► P. falciparum: micro and macrogametocytes are easily recognized by their crescentic, cigar or banana like shape. ► Micro gametocytes have a diffuse chromatin ► while macrogametocytes have thickened chromatin. Mirogametocyte, Giemsa thin smear.

► P.falciparum: in thick films red blood cells are not visible and leucocytes and parasites appear smaller than in thin smears. ► Trophozoites have a ring or comma shape. With one or two dots of chromatin. ► The pigment, when present is compact.

► Plasmodium falciparum: Blood Stage Parasites: Thick Blood Smears

► P. falciparum: trophozoites are small rings with single or double small chromatin dots, and regular cytoplasm; ► multiple infection and high parasitemia (>5%) are common. ► Dots or cleft (Maurer's) can be observed on the infected RBCs.

P.falciparum trophozoites, thin smear, Giemsa stain.

What are the applique forms seen in the blood smear? ► Early ring form of plasmodium falciparum found at the margin of the red cell ► sometimes trophozoites appear at the edge of the red blood cell ( applique form). ► Erythrocytes maintain regular shape and size.

Describe the clinical illness caused by this parasite. Explain the severity of symptoms in this patient. ► The incubation period varies generally between days. ► As the parasite load becomes significant, the patient develops headache, lassitude, vague pains in the bones and joints chilly sensations and fever.

► As the disease progresses, the chills and fever become more prominent. ► The chill and fever follow a cyclic pattern( PAROXYSM) with the symptomatic period lasting 8-12 hours. ► Each paroxysm is due to the rupture of infected erythrocytes and release of parasites.

Why is infection by this species considered to be more serious than infection caused by other species? ► This organism causes sequestration of capillary vasculature in the brain, gastrointestinal and renal tissues. ► Chronic malaria results in splenomegaly, hepatomegaly and nephritic syndromes. ► P falciparum will be will eventually results in death.

► renal failure may result from sequestration of RBCs and alteration of the renal microcirculation. Glomerulal and interstitial vessels present RBCs adhering to the endothelium

► P.falciparum: severe P.falciparum infections are clinical forms characterized by ► potentially fatal manifestations or complications:cerebral malaria, defined by a state of unrousable coma in absence of other causes ► Celebral malaria: parasitized RBCs in brain vessels (H&E stain). most common manifestation.

Describe the life cycle of this parasite? ► Malarial parasites are transmitted by the infected female anopheline mosquito which injects sporozoites present in the saliva of the insect. ► Sporozoites infect the liver paranchymal cells where they may remain dormant or undergo stages of schizogony to produce schizonts and merogony to produce merozoites. ► When parenchymal cells rupture. Thousands of moronts are released into blood and infect the red cells.

► Young schizont with 10 nuclei ► Mature schizont with 24 nuclei, ready to rupture

► P falciparum infects both immature red blood cells and mature red cells. ► In red cells, the parasites mature into trophozoites. ► These trophozoites undergo schizogony and merogony in red cells which ultimately burst and release daughter merozoites.

► Increasingly mature trophozoites

► Some of the merozoites transform into male and female gametocytes while other enter red cells to continue the erythrocytic cycle. ► The gametocytes are ingested by the female mosquito. ► The female gametocyte transforms into ookinete, is fertilized and forms an oocyst in the gut.

► Plasmodium falciparum

► The oocyte produces sporozoites which migrate to the salivary gland and are ready to infect another host. ► Malaria can be tranmitted by transfusion and transplacentally.

In addition to thick and thin blood smears, which other laboratory techniques are available to diagnose the infection ► Malaria diagnosis relies on observation of parasites in Giemsa- stained thin or thick smears (G-TS). ► thin film gives more informations on parasite morphology and permits an easier morphologic differentiation ► Alternative techniques for identification of malaria parasites are based on fluorochromes such as Acridine Orange (AO), DAPI-PI or BCP. Acridine Orange (AO) DAPI-PIAcridine Orange (AO) DAPI-PI ► With these dyes malaria parasites are easily recognized under UV light, reducing the time spent reading the slides.

► P.falciparum: staining with fluorochromes is rapid (less than 1 min) and observation of slides can be performed at low magnification (400X) allowing rapid screening of smears even with low parasitemia.

► quantitative buffy coat (QBC) (Becton-Dickinson) analysis uses AO staining of centrifuged parasites in a capillary tube containing a float, has been shown to be rapid and accurate

► P.falciparum trophozoites. Acridine Orange stain

► P.falciparum: the sensitivity of different isolates of P.falciparum to drugs can be assessed with the WHO "in vitro test". The development to mature schizont in presence of therapeutic levels of the drug demonstrates resistance of the isolate.

How would this infection be treated? ► Treatment is effective with various quinine derivatives( chloroquine, meflaquine and primaquine, etc.) ► Drug resistance, particularly in P. falciparum is a problem. ► Control measures are eradication of infected anopheline mosquitos. ► Vaccines are being developed and tried but none is available yet for routine use.