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FLAGELLATES

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SN 1 - LD Bodies (Leishmania donovani)

Morphology of LD Bodies:

MUHS Exam Tip: LD body = nucleus + rod-shaped kinetoplast inside macrophage. Must differentiate from T. cruzi amastigotes (also inside cells, but form pseudocysts in muscle, not just macrophages).

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SN 2 - Acute Giardiasis: Laboratory Diagnosis and Findings

Clinical Findings in Acute Giardiasis:

• Incubation period: 1-3 weeks
• Acute diarrhea: Watery, foul-smelling, greasy (steatorrhoeic) stools
• Abdominal cramps and bloating
• Flatulence - very characteristic
• Nausea, anorexia
• Self-limited illness lasting 7-10 days
• No blood or mucus in stool (non-invasive parasite)
• Chronic form: malabsorption syndrome (resembling Celiac sprue)

Laboratory Diagnosis:

• Stool examination (wet mount + permanently stained smear)
• Trophozoite form: Seen in acute/diarrheal stage
  • Pear/kite/teardrop shaped
  • Size: 10-20 µm
  • 2 nuclei anteriorly (giving "owl face" appearance)
  • Sucking disc on ventral surface
  • 4 pairs of flagella (8 flagella total)
  • Characteristic "falling leaf" motility on wet mount
• Cyst form: Seen in chronic/formed stool
  • Oval, 8-19 µm
  • 2-4 nuclei
  • Intracytoplasmic fibrils (axonemes) and curved median bodies
  • Cytoplasm retracted from wall
• Concentration methods (formol-ether) increase sensitivity
• Up to 7 stool samples needed for optimal sensitivity (shed sporadically)

• Useful when stool exam is negative
• Trophozoites recovered directly from duodenal fluid

• EIA (ELISA), DFA (Direct Fluorescent Antibody), LFIC rapid tests
• Sensitivity 90-100%, Specificity ~100%
• Faster than microscopy; preferred in resource-rich settings
• Results in < 30 minutes for rapid tests

• PCR-based multiplex stool panels - sensitivity ~100%
• Detects G. duodenalis with high accuracy; also used for genotyping

MUHS Exam Tip: "Falling leaf motility" and "owl face trophozoite" are classic exam picks. Stool examination x3 (minimum) to x7 (optimal) needed.

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LAQ 1 - Leishmania donovani: Life Cycle, Kala-Azar (Visceral Leishmaniasis) - Pathogenicity and Laboratory Diagnosis

Classification

• Kingdom: Protozoa
• Phylum: Sarcomastigophora
• Order: Kinetoplastida
• Class: Flagellates (Hemoflagellates)
• Species: Leishmania donovani

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Life Cycle of Leishmania donovani

1. Invertebrate host (vector): Phlebotomus sandfly (Old World) / Lutzomyia (New World)
2. Vertebrate host: Humans and dogs

Step-by-Step Life Cycle:

1. Sandfly takes a blood meal from an infected human - ingests macrophages containing amastigotes
2. Amastigotes transform into promastigotes in the midgut of the sandfly
3. Promastigotes multiply by binary fission in the gut
4. Migrate anteriorly to the proboscis (infective stage ready in ~1 week)

1. Infected sandfly bites human - promastigotes inoculated into skin
2. Promastigotes immediately phagocytosed by macrophages at the bite site
3. Inside the phagolysosome, promastigotes transform into amastigotes (LD bodies)
4. Amastigotes multiply by binary fission within macrophages
5. Macrophage ruptures → amastigotes infect new macrophages
6. Infected macrophages travel via lymphatics/bloodstream to liver, spleen, bone marrow, lymph nodes (reticuloendothelial system)
7. Cycle continues → tissue destruction and clinical disease

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Kala-Azar (Visceral Leishmaniasis) - Pathogenicity

Pathogenesis:

1. Massive multiplication of amastigotes in macrophages throughout the RES
2. Splenomegaly - due to hyperplasia of macrophages packed with LD bodies; spleen becomes the largest solid organ in the body
3. Hepatomegaly - Kupffer cell hyperplasia with amastigotes
4. Bone marrow infiltration - replacement of normal marrow leads to:
   • Anaemia (normocytic normochromic)
   • Leucopenia
   • Thrombocytopenia - i.e., Pancytopenia
5. Hypergammaglobulinemia - polyclonal B-cell activation (non-specific antibodies - NOT protective)
6. Hypoalbuminemia - due to malnutrition and liver involvement → edema
7. Aldehyde test (Napier's) becomes positive due to elevated serum globulins
8. Skin darkening (hyperpigmentation) - especially on face, hands, feet - due to increased melanin; giving the name "black fever"
9. Post-Kala-Azar Dermal Leishmaniasis (PKDL) - hypopigmented or erythematous skin lesions appear months-years after apparently successful treatment

Clinical Features of Kala-Azar:

• Prolonged irregular fever ("double-daily rise" pattern)
• Massive splenomegaly (most important sign) - can reach the pelvis
• Hepatomegaly
• Progressive weight loss and cachexia
• Anaemia - pallor
• Skin darkening
• Lymphadenopathy (in African form)
• Hypergammaglobulinemia

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Laboratory Diagnosis of Kala-Azar

• Stain: Giemsa (preferred), Leishman, H&E
• Look for: LD bodies (oval amastigotes with nucleus + kinetoplast) inside macrophages

• PCR on peripheral blood, bone marrow, or tissue - highest sensitivity; used in reference labs

• Negative in active kala-azar (due to depressed CMI)
• Becomes positive only after successful treatment
• NOT useful for diagnosis

MUHS Exam High-Yield Points:

• Splenic aspirate: highest sensitivity (>95%) but risky
• Bone marrow biopsy: safest definitive diagnosis
• rK39 ICT: used in national Kala-Azar elimination program
• Aldehyde test = positive in kala-azar (nonspecific)
• Leishmanin test = NEGATIVE in active disease
• Treatment: Liposomal Amphotericin B (single dose 10 mg/kg IV) - used in India's national program; also Miltefosine (oral)

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SPOROZOA

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SN 1 - Malaria: Laboratory Diagnosis

Laboratory Diagnosis of Malaria

1. Thick film - concentration method; detects presence of parasite (screening); better sensitivity at low parasitemia
2. Thin film - species identification; morphology better appreciated

MUHS Key Diagnostic Smear Features:

• P. falciparum: Multiple rings + accolé position + banana gametocytes
• P. vivax: Enlarged RBC + Schüffner dots + amoeboid trophozoite
• P. malariae: Rosette schizont + band/bar trophozoite forms

• Immunochromatographic lateral flow strips
• Use monoclonal antibodies against:
  • HRP-2 (Histidine-Rich Protein 2) - P. falciparum specific
  • pLDH (Plasmodium Lactate Dehydrogenase) - pan-malarial
  • Aldolase - pan-malarial
• Results in <20 minutes
• P. falciparum RDTs: sensitivity 94-100%, specificity 94.2%
• BinaxNOW (FDA approved) detects HRP-2 and pLDH

• PCR (most sensitive; 18S rRNA gene target) - species differentiation, drug resistance markers
• LAMP (Loop-Mediated Isothermal Amplification) - field-deployable
• NASBA (Nucleic Acid Sequence-Based Amplification)

• Not for acute diagnosis; used for epidemiological surveys and blood donor screening
• Remains positive ~1 year after treatment

• Anaemia (normocytic normochromic) - haemolysis
• Thrombocytopenia - very common in malaria
• Elevated LDH, bilirubin
• Leucopenia

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SN 2 - Plasmodium falciparum Infections: Complications and Laboratory Diagnosis

Plasmodium falciparum - Overview

• Causes Malignant Tertian Malaria
• Most dangerous species; responsible for majority of malaria deaths
• Incubation period: 7-10 days (shortest of all species)
• No hypnozoites - no relapses from liver (only recrudescence)
• Distribution: tropical and subtropical regions

Complications of P. falciparum Malaria (★★★ MUHS FAVOURITE)

1. Infects ALL ages of RBCs (not just young or old) → massive parasitemia
2. Cytoadherence: Infected RBCs adhere to vascular endothelium (via PfEMP1 - P. falciparum Erythrocyte Membrane Protein 1), forming "knobs" on RBC surface
3. Rosetting: Infected RBCs bind to uninfected RBCs
4. Capillary plugging: Masses of parasitized RBCs, platelets, leucocytes and malarial pigment block capillaries
5. Sequestration of schizonts and trophozoites in deep capillaries → rarely seen in peripheral blood

Complications:

WHO Criteria for Severe Malaria: Impaired consciousness, >5% parasitemia, severe anaemia (Hb <7 g/dL), renal failure, pulmonary oedema, hypoglycaemia, shock, bleeding, hyperparasitemia, jaundice, repeated convulsions.

Laboratory Diagnosis of P. falciparum:

• Multiple ring forms within a single RBC (2 or more rings/cell)
• Rings in accolé (appliqué) position - ring appears at the periphery/margin of RBC
• No enlargement of RBC
• Schizonts and trophozoites usually absent (sequestered in deep capillaries)
• Crescentic (banana/sausage-shaped) gametocytes = PATHOGNOMONIC of P. falciparum
• Maurer dots (dots/clefts) occasionally seen
• High parasitemia (>10%) of ring forms = strongly suggestive even without gametocytes

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LAQ 1 - Malignant Tertian Malaria: Life Cycle, Complications, Laboratory Diagnosis

Life Cycle of Plasmodium Species (Common to All 5 Species)

• Definitive host: Female Anopheles mosquito (sexual reproduction occurs here)
• Intermediate/Incidental host: Humans (asexual reproduction)

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Phase 1: EXOERYTHROCYTIC (Pre-erythrocytic) Cycle - in Liver

1. Infected female Anopheles mosquito bites human → inoculates sporozoites via saliva
2. Sporozoites enter bloodstream → travel to liver parenchymal cells within 30 minutes
3. In hepatocytes: sporozoites undergo schizogony (asexual division) → liver schizont
4. Liver schizont matures → 10,000-40,000 merozoites released
5. Hepatocytes rupture → merozoites enter bloodstream
6. Duration: 7-10 days for P. falciparum; 8-25 days depending on species

• P. vivax and P. ovale can form HYPNOZOITES (dormant forms in liver) → cause RELAPSE months to years later
• P. falciparum and P. malariae do NOT form hypnozoites (recrudescence, not relapse)

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Phase 2: ERYTHROCYTIC Cycle - in RBCs

1. Merozoites attach to specific receptors on RBC surface → enter RBC
2. Ring stage (early trophozoite): ring appearance with hemozoin pigment forming
3. Growing trophozoite stage: fills RBC, pigment granules appear
4. Schizont stage: nucleus divides → merozoites formed (species-specific number)
5. RBC ruptures → merozoites released into bloodstream → infect new RBCs → FEVER PAROXYSM
6. Duration of erythrocytic cycle:
   • P. falciparum, P. vivax, P. ovale: 48 hours → Tertian (fever every 48 hours, day 1 and day 3)
   • P. malariae: 72 hours → Quartan (fever every 72 hours)
7. Some merozoites develop into male (microgametocyte) and female (macrogametocyte) in RBCs

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Phase 3: SEXUAL Cycle (Sporogony) - in Mosquito

1. Female Anopheles bites infected human → ingests blood containing mature gametocytes
2. In mosquito midgut:
   • Exflagellation of microgametocyte → 8 motile microgametes
   • Macrogametocyte → macrogamete
3. Fertilization → Zygote (non-motile)
4. Zygote → elongated Ookinete (motile)
5. Ookinete penetrates gut wall → forms Oocyst on outer gut wall
6. Oocyst matures → contains sporozoites (10,000+)
7. Oocyst ruptures → sporozoites released → migrate to salivary glands
8. Ready to infect next human host
9. Duration in mosquito: ~2 weeks (10-14 days at 27°C)

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Malignant Tertian Malaria - Key Points:

• Caused by P. falciparum
• "Malignant" - because it is life-threatening
• "Tertian" - fever spikes every 48 hours (days 1 and 3)
• Complications listed in full in SN 2 above

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LAQ 2 - Plasmodium vivax: Morphology, Life Cycle, Laboratory Diagnosis

Plasmodium vivax - Overview

• Causes Benign Tertian Malaria
• Most prevalent plasmodium species with widest geographic distribution
• Predominantly in South America and Southeast Asia
• Invades only young, immature RBCs (reticulocytes)
• Receptor: Duffy blood group antigen on RBC surface (Duffy-negative individuals (West Africans) are naturally resistant, though rare cases now reported)
• Unique feature: Forms hypnozoites in the liver → RELAPSE

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Morphology of P. vivax

MUHS Mnemonic for P. vivax: "SAVES" - Schüffner dots, Amoeboid trophozoite, Vivid enlargement of RBC, Extra merozoites (up to 24), Sleeping forms (hypnozoites)

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Life Cycle of P. vivax

• Exoerythrocytic phase: 8 days (some sporozoites become HYPNOZOITES)
• Hypnozoites: Dormant liver stage; can reactivate after months to years → RELAPSE
• Erythrocytic cycle: 48 hours → Tertian fever (every 48 hours)
• Schizont: Up to 24 merozoites
• Gametocytes: Rounded, fill enlarged RBC

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Clinical Features of P. vivax Malaria:

• Incubation period: 10-17 days (short incubation); can be months if hypnozoites
• Prodrome: Vague flu-like illness - headache, myalgia, photophobia, anorexia
• Classic Malarial Paroxysm (every 48 hours - tertian pattern):
  • Cold stage: Chills, rigors (shivering) - 15 min to 1 hour
  • Hot stage: High fever 103-106°F, headache, vomiting - 2-6 hours
  • Sweating stage: Profuse sweating, fever drops, exhaustion - 2-4 hours
• Splenomegaly - progressive
• Anaemia - progressive haemolytic
• Recent evidence: P. vivax can also cause severe disease - pulmonary oedema, renal failure, cerebral involvement (not just P. falciparum)

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Laboratory Diagnosis of P. vivax:

• Thick film: Screening; detect presence of parasites
• Thin film: Species identification (Giemsa/Leishman stain)
• Characteristic features:
  • Enlarged RBC with Schüffner dots (pink granules)
  • Amoeboid growing trophozoite
  • Schizont with up to 24 merozoites
  • Rounded gametocyte filling enlarged RBC
• Best time: Between paroxysms (4-6 hours after fever) when peak parasitemia

• Anti-pLDH (pan-malarial) and anti-aldolase antibody tests
• Specific P. vivax pLDH-based tests available
• Sensitivity 67-86% for P. vivax (lower than for falciparum)
• Not recommended as sole diagnostic test for P. vivax in low-endemic settings

• IFAT, ELISA - used for epidemiological studies, not acute diagnosis
• Cross-react with other Plasmodium species

• Highest sensitivity for P. vivax
• Used for mixed infections and species confirmation
• Detects P. vivax at very low parasitemia (sub-microscopic infections)

• Anaemia (normocytic normochromic)
• Thrombocytopenia
• Elevated bilirubin (indirect/unconjugated)
• Elevated LDH
• Leucopenia

MUHS TREATMENT TIPS:

• Chloroquine: For blood-stage P. vivax (in sensitive areas)
• Primaquine: Essential to eliminate hypnozoites and prevent relapse; check G6PD before giving (causes haemolysis in G6PD-deficient patients)

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QUICK COMPARISON TABLE - All Plasmodium Species (MUHS EXAM FAVOURITE)

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KEY EXAM MNEMONICS

• Foul-smelling greasy stool
• Acute diarrhea (self-limited 7-10 days)
• Trophozoites with "falling leaf" motility
• Malabsorption in chronic form
• Antigenic detection by ELISA
• No blood/mucus in stool

• Blackwater fever
• Encephalopathy (Cerebral malaria)
• ARDS / pulmonary oedema
• Coagulopathy (DIC)
• Hypoglycaemia
• Hyperparasitemia
• Anaemia (severe)
• Renal failure
• Pancytopenia

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