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Standard Tests for Diagnosis of Malaria

Key principle: Malaria is NOT a clinical diagnosis. All fever cases in or returning from a malaria-endemic area must be confirmed by a laboratory test. - Harrison's Principles of Internal Medicine, 22E

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1. Blood Smear Microscopy (Gold Standard)

Thin Blood Smear

• Air-dried, fixed in anhydrous methanol, then stained
• Examined under oil immersion (x1000 magnification)
• Used to identify the Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale, P. knowlesi) and determine parasitemia (number of parasitized RBCs per 1000 RBCs)

Thick Blood Smear

• NOT fixed before staining (RBCs lyse during staining)
• Concentrates parasites 40-100x compared to a thin smear - much higher sensitivity for detecting low-density infections
• Parasites and WBCs are counted together; parasitemia is then calculated
• A minimum of 200 WBCs must be counted under oil immersion before calling a smear negative
• 100-200 fields must be examined before declaring a thick smear negative

Prognostic indicators on smear:

• >10^5 parasites/μL = increased risk of death
• >20% of parasites with visible pigment = poor prognosis in severe malaria
• Phagocytosed malarial pigment in >5% of neutrophils = recent schizogony, poor prognosis

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2. Rapid Diagnostic Tests (RDTs)

Antigens detected:

Types:

• Monovalent RDT - detects only P. falciparum
• Bivalent RDT - carries a second antibody (pan-malaria or P. vivax-specific), distinguishing falciparum from non-falciparum malaria

Important caveats:

• PfHRP2-based RDTs can remain positive for several weeks after treatment (helpful in severe malaria after drug clearance, but misleading in high-transmission areas)
• RDTs cannot quantify parasitemia - a key limitation
• PfHRP2/3 gene deletions in some P. falciparum strains (especially in the Horn of Africa) cause false-negative results with PfHRP2-based RDTs - a growing problem

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3. Molecular Diagnosis - PCR

• More sensitive than both microscopy and RDTs
• Accurately defines malaria species, including mixed infections
• Detects submicroscopic parasitemia
• Used in reference centers for confirmation, species typing, drug resistance genotyping, and epidemiological studies
• NOT used for primary diagnosis in endemic settings due to cost, infrastructure needs, and time required

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4. Fluorescence Microscopy (Acridine Orange Staining)

• Staining parasites with the fluorescent dye acridine orange allows rapid diagnosis in patients with low-level parasitemia
• Limitation: does not allow speciation - cannot identify which Plasmodium species is present
• Used in field settings where it is faster than conventional Giemsa microscopy

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5. Serology (Antibody Tests)

• Detects antibodies against malaria rather than the parasite itself
• Not useful for diagnosing acute infection (antibodies appear late and persist long after infection)
• Used mainly for epidemiological surveys and blood donor screening
• Has no role in clinical diagnosis of acute malaria

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Summary Table

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Clinical Approach (per WHO/National Guidelines)

1. Where microscopy result is available within 24 hours: Treat based on species identified on smear (P. falciparum vs P. vivax)
2. Where microscopy is unavailable and monovalent RDT is used: Treat based on RDT result; if positive, presume P. falciparum
3. Where bivalent RDT is used: Distinguishes falciparum from vivax, guides appropriate drug choice (ACT vs chloroquine + primaquine)

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Standard Tests for Malaria Diagnosis

1. Blood Smear Microscopy (Gold Standard)

• Thick smear - higher sensitivity, concentrates parasites 40-100x, used for detection
• Thin smear - used for species identification and parasitemia quantification
• Stained with Giemsa stain (preferred), or Wright's/Leishman's/Field's stain
• Must examine 100-200 fields before calling a smear negative

2. Rapid Diagnostic Tests (RDTs)

• Detect parasite antigens (PfHRP2, pLDH, aldolase) from a finger-prick sample
• Monovalent - detects P. falciparum only
• Bivalent - distinguishes P. falciparum from P. vivax
• Cannot quantify parasitemia; PfHRP2 tests may miss strains with gene deletions

3. PCR (Molecular Diagnosis)

• Most sensitive and specific
• Identifies all species and mixed infections
• Used in reference labs only - not for routine field diagnosis

4. Acridine Orange Fluorescence Microscopy

• Rapid, good for low-density parasitemia
• Cannot speciate the parasite

5. Serology (Antibody Tests)

• Detects past infection, not active disease
• Used for epidemiology and blood bank screening only - no role in acute diagnosis

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Key rule: Malaria is never a clinical diagnosis alone - always confirm with a lab test. Scroll up to see the full detailed breakdown with images and treatment flow charts.

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Diagnostic Tests for Anemia

Anemia is defined as hemoglobin below normal for a given population. The WHO thresholds are: <13 g/dL in men, <12 g/dL in non-pregnant women, <11 g/dL in pregnant women.

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Step 1: Complete Blood Count (CBC) - First-Line Test

Red Cell Indices (Table 66-1, Harrison's 22E)

MCV-Based Classification (Most Practical Approach)

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Step 2: Peripheral Blood Smear - Mandatory for All Anemias

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Step 3: Reticulocyte Count - Assesses Bone Marrow Response

• Corrected reticulocyte count = Measured % × (Patient Hct / 45)
• Absolute reticulocyte count (preferred): directly measures reticulocytes per volume - no correction needed

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Step 4: Iron Studies - For Microcytic or Suspected Iron-Deficiency Anemia

Serum ferritin is the single best test for iron deficiency: a ferritin <15 ng/mL virtually confirms iron deficiency (high specificity); ferritin >100 ng/mL has a very low likelihood ratio for iron deficiency (LR- ~0.08). - Symptom to Diagnosis, 4th Ed.

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Step 5: Additional Targeted Tests (Based on Suspected Cause)

For Macrocytic / Megaloblastic Anemia

• Serum Vitamin B12 - <200 pg/mL suggests deficiency
• Serum folate / RBC folate - RBC folate is more reliable
• Methylmalonic acid (MMA) and Homocysteine - elevated in B12 deficiency; only homocysteine elevated in folate deficiency
• Anti-intrinsic factor antibodies - for pernicious anemia
• Anti-parietal cell antibodies - for pernicious anemia

For Hemolytic Anemia

• LDH - elevated (released from lysed RBCs)
• Indirect bilirubin - elevated (from heme breakdown)
• Haptoglobin - decreased (consumed by free hemoglobin)
• Direct Coombs Test (DAT) - positive in autoimmune hemolytic anemia
• Urine hemoglobin / hemosiderinuria - intravascular hemolysis

For Suspected Hemoglobinopathy / Thalassemia

• Hemoglobin electrophoresis - identifies abnormal Hgb variants (HbS, HbC, HbH)
• HPLC (High-Performance Liquid Chromatography) - gold standard for Hgb typing
• Genetic/molecular testing - for alpha-thalassemia gene deletions

For Aplastic Anemia / Bone Marrow Failure

• Bone marrow aspiration + biopsy - mandatory
  • Normal M:E ratio = 2.5:1
  • Performed from the posterior iliac spine
  • Biopsy must be done BEFORE aspiration to avoid artifact
  • Indications include: circulating blasts, severe pancytopenia, reticulocytes <0.1%, unexplained severe anemia, evidence of marrow infiltration

For Renal Anemia

• Serum creatinine / eGFR - chronic kidney disease suppresses EPO
• Serum EPO level - inappropriately low in renal anemia

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Diagnostic Algorithm Summary

ANEMIA CONFIRMED (low Hgb on CBC)
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    Peripheral Blood Smear + Reticulocyte Count
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    |                |                             |
MICROCYTIC        NORMOCYTIC                  MACROCYTIC
(MCV <80)         (MCV 80-100)               (MCV >100)
    |                |                             |
Iron studies    Reticulocyte count          B12, folate, MMA
Ferritin         High → Hemolysis/          Homocysteine
TIBC              blood loss               Thyroid function
Hgb electro-     Low → Marrow failure/      Liver enzymes
phoresis          CKD/chronic disease       Bone marrow biopsy

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