In a patient with Plasmodium vivax infection who had a negative glucose‑6‑phosphate dehydrogenase (G6PD) test and now presents with jaundice and anemia after discontinuing primaquine, what test should be ordered to evaluate for hemolytic anemia?

Diagnostic Workup for Hemolytic Anemia in Vivax Malaria with Suspected G6PD Deficiency

You must immediately repeat G6PD testing using a quantitative assay, as the initial "negative" test was likely falsely reassuring, and then obtain a complete hemolytic workup including reticulocyte count, peripheral blood smear, lactate dehydrogenase (LDH), haptoglobin, indirect bilirubin, and direct antiglobulin test (DAT).

Critical First Step: Retest G6PD Status

The most important test is a quantitative G6PD enzyme activity assay, as qualitative screening tests have significant limitations, particularly in females with intermediate deficiency and can miss clinically significant variants. 1

• Quantitative G6PD testing is essential because the initial negative test may have been:

  • A qualitative test with poor sensitivity for intermediate deficiency 2
  • Performed during acute hemolysis when reticulocytes (which have higher G6PD activity) falsely elevate results 3
  • Unable to detect heterozygous females with mosaic enzyme expression 2

• The Mediterranean G6PD variant (B-) and certain Asian variants carry very high risk of severe complications with primaquine, making accurate diagnosis critical for future treatment decisions. 1

• Samples should ideally be stored at 4°C and tested within 4 days of collection to preserve enzyme activity. 4

Essential Hemolytic Workup

Order the following tests to confirm and characterize hemolytic anemia:

• Reticulocyte count: This will be elevated in hemolysis and helps distinguish hemolytic from other causes of anemia; expect peak elevation 7-14 days after primaquine exposure in G6PD-deficient patients. 5

• Peripheral blood smear: Look for bite cells, blister cells, and Heinz bodies (with supravital staining) characteristic of oxidative hemolysis from primaquine in G6PD deficiency.

• Lactate dehydrogenase (LDH): Elevated in hemolysis due to release from lysed red blood cells.

• Haptoglobin: Will be decreased or absent in intravascular hemolysis.

• Indirect (unconjugated) bilirubin: Elevated in hemolysis, explaining the jaundice; this distinguishes hemolytic jaundice from hepatic causes.

• Direct antiglobulin test (DAT/Coombs): Should be negative in G6PD-related hemolysis, helping exclude autoimmune hemolytic anemia.

Additional Considerations

Screen for hemoglobinopathies (hemoglobin electrophoresis) as HbE and thalassemia are common in Southeast Asian populations and influence hemolytic severity. 5

• In one Cambodian study, 26 patients had HbE and 6 had α-/β-thalassemia, with thalassemia significantly influencing the initial decrease in hemoglobin (P = 0.027). 5

• These conditions can coexist with G6PD deficiency and compound anemia risk.

Monitor hemoglobin trends closely: The greatest hemoglobin drop typically occurs within the first week after primaquine exposure, with mean decreases of -1.88 g/dL (range -5.8 to 0 g/dL) in one study. 5

Common Pitfalls to Avoid

• Do not rely on a single qualitative G6PD test: Qualitative tests have 100% negative predictive value for severe deficiency in males but perform poorly in heterozygous females (sensitivity only 83%). 2

• Do not test G6PD during acute hemolysis: Young reticulocytes have higher enzyme activity and can mask deficiency; ideally retest 2-3 months after the acute episode if immediate quantitative testing is unavailable.

• Do not assume hepatitis A reactivation: Anti-HAV IgG reflects past resolved infection and does not indicate active disease; hepatitis A does not reactivate in immunocompetent individuals. 1