ASM Clinical Core Curriculum II More Pitfalls in the Morphologic Diagnosis Blood-borne Parasites - PDF

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ASM Clinical Core Curriculum II More Pitfalls in the Morphologic Diagnosis Blood-borne Parasites Julie Ribes, MD, PhD Bobbi Pritt, MD Objectives Upon completion
ASM Clinical Core Curriculum II More Pitfalls in the Morphologic Diagnosis Blood-borne Parasites Julie Ribes, MD, PhD Bobbi Pritt, MD Objectives Upon completion of this session, participants should be able to: Describe common pitfalls in the morphologic diagnosis of malaria and other infectious agents in blood films Disclosures: None Demonstration and Identification of Blood Parasites - General Giemsa-stained thick and thin blood films are the gold standard for diagnosis of malaria and other blood parasites Other methods: Antigen detection, PCR, serology Concentration methods for microfilariae and trypanosomes Malaria Plasmodium speciation Based on simultaneous examination of numerous characteristics: Size of infected RBC Presence/absence of RBC stippling and inclusions Life cycle stages present So Many Features Where do I start? What are the Stages? Amoeboid trophozoite of P. vivax Basket form and Band form of P. malariae Early-stage trophozoites rings Late-stage trophozoites Schizonts Gametocytes Size of Infected RBCs Normal or small Enlarged P. falciparum (normal RBCs) or P. malariae (norm or small RBCs) No stippling; Maurer's clefts in P. falciparum Relevant to most stages of the parasite! P. vivax or P. ovale Supporting feature: Stippling P. falciparum P. malariae RINGS AND ALL STAGES PRESENT GAMETOCYTES Thick rings PREDOMINATE 1/3 size RBC Small delicate rings Basket, band forms 1/3 size RBC Rosette schizonts with Headphones forms 8-10 merozoites Applique forms Multiple rings/rbc High parasitemia Banana-shaped gametocytes P. vivax ALL STAGES PRESENT Amoeboid trophs 1/3 size RBC Schizont with merozoites P. ovale ALL STAGES PRESENT Trophs more compact 1/3 size RBC 1/3 oval shape fimbriated edges Which Species am I? Blood Answer: Parasites P. falciparum (trophozoites) Blood Answer: Parasites P. malariae (trophozoite basket form) Answer: P. vivax (trophozoites) P. vivax (trophozoites) P. ovale gametocytes with pale RBC cytoplasm Other blood parasites seen on peripheral blood smears Trypanosomes Microfilariae Diagnostic Pitfalls Morphologic Diagnosis Morphological diagnosis of blood parasites is effected by. Stain solutions ph Anticoagulants Anti-malarial treatment Experience Our focus for today Case #1 Case #1: An Adoptee from Ethiopia 4-year-old black international adoptee who presented with muscle weakness and encephalopathy Work up is routine, but may also be based on symptoms Laboratory Test Patient s Test Result CBC HCT = 30.5%(nl 33-43%) RBC = 4.46m/uL(nl m/uL) MCV = 68fL (nl 75-95fL) RDW = 18 (nl ) Slight Poikilocytosis Slight Polychromatophilia Chemistries and LFT s HIV and Hepatitis panel Stool O&P x 3 Sickle-Dex Normal Normal No Ova or Parasites detected Positive for Hgb S Case #1: An Adoptee from Ethiopia Malaria examination x 1 is generally ordered Thick film Thin film Interpretive reports must state that a single negative result is not adequate to rule out malarial infection Reticulocyte count advisable Markers of hemolysis (bilirubin, haptoglobin) advisable How would you sign this case out? 1. Plasmodium malariae trophozoite 2. Plasmodium falciparum trophozoite 3. Plasmodium spp. merozoite 4. No blood parasites identified Platelets and Howell-Jolly bodies present Blood Howell-Jolly Parasites Bodies P. falciparum rings Platelets Howell-Jolly body Platelets Platelets P. falciparum rings Case #1 An Adoptee from Ethiopia Peripheral blood film findings suggested functional asplenia: NRBC and Howell Jolly Bodies Increased poikilocytosis (including sickle cells and target cells and anisocytosis? Secondary to Sickle Cell Anemia with autosplenectomy no history of this Hemoglobin electrophoresis was performed due to microcytosis and positive Sickle-Dex 61% Hgb A / 35% Hgb S / 3.5% Hgb A 2 Suggests Hgb S and alpha thalassemia Influence of Hemoglobinopathies and RBC Antigens on Malarial Infections Hbg S offers partial protection from malaria ~8% of African Americans and ~25% of blacks in Sub-Saharan Africa have Hgb S trait Hgb S trait individuals have a high rate of survival Sickle disease patients, however, have high mortality with malarial infections Other hemoglobinopathies and hereditary hemolytic anemias offer no protective advantages Duffy antigens serve as the receptor for both P. vivax and P. knowlesi P. vivax is relatively absent from West Africa where 95% of the black population is Duffy negative Case #2 CAP Proficiency Challenge Case History: CAP proficiency specimen was received in the lab The slide was read by the tech who felt that a mixed infection was present: P. vivax P. falciparum (gametocytes only) What would you call this case? 1. Plasmodium falciparum 2. Plasmodium vivax 3. Plasmodium ovale 4. Plasmodium malariae 5. Mixed infection with P. falciparum and P. vivax All morphologies can be attributed to a single species Morphology is altered when reading in areas of the slide that are too thick or too thin This technologist wrongly attributed altered gametocytes as banana-shaped gametocytes of P. falciparum What area of the slide is best for morphology? Too thin Ideal Too thick Back to this case Amoeboid trophozoites In enlarged RBCs with Faint stippling is Consistent with P. vivax Note that the trophozoite Cytoplasm is beginning to Look banana-like but these are NOT real gametocytes of P. falciparum In thick areas, cytoplasm is not easily visible Note similar appearing rounded and elongate forms In the same field No brown malarial pigment seen not a gametocyte Note brown malarial pigment with the P. falciparum gametocyte Mimics of P. falciparum gametocytes True P. falciparum gametocytes Take Home Messages Morphology can be misinterpreted when reading in non-ideal portions of the slide Mixed infections can happen, so it is a good idea to look out for them However, it is important to identify 2 or more Distinct morphologic populations before calling a mixed infection Case #3 A 15-year-old boy from Brazil with fever, tachycardia and mild chest discomfort Another CAP proficiency challenge the most common source of positive specimens for many labs 10 um 5 µm Diagnosis? 1. Plasmodium rings, favor P. falciparum 2. Plasmodium rings, favor P. malariae 3. Plasmodium rings, favor P. vivax 4. Microfilariae present 5. Trypanosomes present Take Home Message from this Case Measuring up may be useful Ocular micrometer should be available for individuals reading malarial films Compare sizes with RBC (~ 7 µm diameter) for thin films Correlate carefully thick and thin films 300 fields need to be reviewed for both thick and thin films for a case (may use multiple slides per case to assure best reading morphology target areas) Note well the location of the parasites with regards to the RBC (intra- or extracellular) Case #4 Cyclic Fevers for 18 Months Following a Trip to Mexico 42 year old male with an 18 month history of fever, and more recently a rash Illness started after returning from a 6-week missionary trip to Acapulco and the surrounding rural mountain-sides He described fevers of that occurred 1-2 times per week together with fatigue and insomnia, lasting 2 day each time Three to four times each week he experienced paresthesias in his legs -Some events were associated with diarrhea Two weeks prior to evaluation, the patient reported development of a very high fever associated with a patchy rash that the wife said looked just like the internet pictures of RMSF - He was visiting the Arkansas/Missouri border on business (but he was already feeling unwell before he left on the trip) Evaluation by his primary care physician was not diagnostic, so he was referred to ID A peripheral blood smear for parasite examination was performed At the point of inoculation of 1 of 4 slides something was seen.. Head Space Nuclei are actually RBCs in rouleaux formation Steps to Examination for Microfilariae: Step 1: Scan ALL slides (thick and thin) at 10x, including the feathered edge and thicker areas Step 2: Look for identifiable features: head, tail, internal nuclei, sheath Step 3: Identification to genus/species level if possible Sheath? Sheathed Wuchereria bancrofti Loa loa Brugia spp. Unsheathed Mansonella perstans Mansonella ozzardi Mansonella streptocerca Onchocerca volvulus Skin snips Wears Long Britches Other supportive features: length, tail nuclei (presence/absence and spacing), head space, staining of sheath, tail hook Pitfalls in microfilaria identification Worms that have lost their sheath CAP Proficiency challenge Something here? Differentiation of sheathed microfilariae Notice pink staining sheath.. Wuchereria bancrofti Loa loa Brugia spp. Take Home Messages The first step to identification of microfilariae is to determine if a sheath is present Microfilariae can lose their sheath! It is essential to examine all microfilariae on the slide for presence of a sheath and other morphologic features. Looking for More Practice?
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