G6PD Deficiency
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G6PD (glucose-6-phosphate dehydrogenase) deficiency is the most common enzymatic disorder of red blood cells worldwide, affecting approximately 400 million people with variable prevalence across populations. The condition results from mutations in the X-linked G6PD gene, leading to reduced activity of the G6PD enzyme, which normally protects erythrocytes from oxidative stress by maintaining reduced glutathione (GSH). Clinical manifestations range from asymptomatic carrier states to life-threatening hemolytic crises, typically triggered by oxidative stressors including medications (sulfonamides, antimalarials, NSAIDs), infections, fava bean ingestion, and other oxidative challenges. The prevalence is highest in populations from Africa, the Mediterranean region, and Southeast Asia, where the trait may confer protection against malaria. Understanding G6PD deficiency is critical for USMLE Step 2 CK because it appears frequently in clinical vignettes involving hemolytic anemia, drug interactions, and ethnic-specific medicine, and because missing the diagnosis can lead to iatrogenic severe hemolysis.
Overview of Normal G6PD Function
The G6PD enzyme catalyzes the first step of the pentose phosphate pathway, converting glucose-6-phosphate to 6-phosphogluconolactone while reducing NADP+ to NADPH. NADPH is essential for maintaining reduced glutathione (GSH), which protects the erythrocyte against oxidative damage by serving as a substrate for glutathione peroxidase, the enzyme that detoxifies hydrogen peroxide and organic peroxides. In normal red blood cells, this antioxidant defense system continuously neutralizes reactive oxygen species (ROS) generated during normal aerobic metabolism and increased production during infection or exposure to oxidative drugs.
Key Mechanism 1: Impaired Pentose Phosphate Pathway and GSH Depletion
In G6PD deficiency, reduced or absent enzyme activity severely compromises the production of NADPH. Without adequate NADPH, glutathione reductase cannot convert oxidized glutathione (GSSG) back to its reduced form (GSH), leading to progressive GSH depletion within erythrocytes. As GSH levels fall below critical thresholds, the red blood cell loses its capacity to neutralize oxidative stressors. This creates a paradoxical situation where the RBC is simultaneously exposed to oxidative challenge (either endogenous from infection or exogenous from drugs) and lacks the biochemical machinery to defend itself. The result is uncontrolled accumulation of ROS, which damages the lipid bilayer, proteins, and hemoglobin itself through oxidative cross-linking.
Key Mechanism 2: Hemoglobin Oxidation and Heinz Body Formation
Excessive ROS precipitates hemoglobin into insoluble aggregates called Heinz bodies (denatured hemoglobin precipitates). These appear as electron-dense inclusions on electron microscopy or as small inclusions on supravital staining (crystal violet or brilliant cresyl blue). Heinz bodies are not seen on standard Wright-Giemsa staining because they are removed by splenic macrophages during passage through the spleen—a process called "pitting." As the spleen removes Heinz bodies, it damages the RBC membrane, creating a partially intact cell with a bite-like defect (hence the term "bite cells"), which is a characteristic though not pathognomonic finding on blood smear.
Key Mechanism 3: Acute Hemolytic Crisis—Membrane Damage and RBC Destruction
Beyond Heinz body formation, ROS causes direct oxidative damage to the RBC membrane through lipid peroxidation and protein cross-linking. This destabilizes the membrane structure and activates abnormal phosphatidylserine exposure on the RBC surface, triggering recognition by splenic macrophages and leading to extravascular hemolysis (destruction within the spleen and liver). The severity of hemolysis depends on the magnitude of the oxidative insult and the degree of G6PD enzyme deficiency. Acute hemolytic episodes typically peak within 24-48 hours of exposure to an inciting agent and can self-limit even with continued exposure because reticulocytes (young RBCs) have higher G6PD activity than mature RBCs, gradually replacing the damaged older cell population.
Key Mechanism 4: Genetic Heterogeneity and Variable Enzyme Activity
Over 160 different G6PD mutations have been identified. These mutations produce enzymes with varying degrees of residual activity, classified into five phenotypic classes by the World Health Organization. Class I mutations result in severe deficiency (<10% normal enzyme activity) with chronic hemolysis; Class II mutations cause severe deficiency with hemolysis only upon oxidative stress; Class III mutations produce mild deficiency (10-50% activity); Class IV mutations result in normal or near-normal activity; and Class V mutations produce increased enzyme activity. The vast majority of clinically significant disease involves Class I or II variants. Additionally, because the G6PD gene is X-linked, males hemizygous for the mutation express the phenotype fully, while heterozygous females may exhibit variable expression depending on X-inactivation patterns (lyonization), explaining why some female carriers can have severe hemolytic crises while others remain asymptomatic.
Key Mechanism 5: Infection-Induced Oxidative Stress
Infections (bacterial, viral, or rarely parasitic) trigger hemolysis in G6PD deficiency through multiple mechanisms independent of specific medications. Activated neutrophils and macrophages generate ROS as part of the innate immune response to pathogens (oxidative burst). Additionally, certain pathogens such as Plasmodium species directly generate ROS through their metabolic activities. Interestingly, the high prevalence of G6PD deficiency in malaria-endemic regions suggests evolutionary selection pressure, as G6PD deficiency paradoxically provides partial protection against severe Plasmodium falciparum malaria—a classic example of balanced polymorphism where the heterozygote advantage (protection against malaria) outweighs the disadvantage of occasional hemolytic crises.
Primary Cause: Mutations in the X-linked G6PD Gene
The G6PD gene is located on the X chromosome (Xq28), and mutations causing loss or reduction of enzyme function are inherited in an X-linked pattern. Hemizygous males (one mutant allele) typically express full clinical phenotype, while heterozygous females may be asymptomatic or mildly affected due to random X-inactivation. The most common variants worldwide include the African A- variant (common in sub-Saharan Africa and African diaspora, Class II, ~10% activity), the Mediterranean variant (common in Greece, Italy, and Middle Eastern populations, Class II, variable activity), and the Asian A variant (common in Southeast Asia). These represent natural population-level genetic variation rather than new mutations in most affected individuals.
Oxidative Drug Triggers (Class A Drugs—High Risk)
These medications have strong and consistent association with hemolysis in G6PD-deficient individuals:
- Antimalarials: Primaquine (the prototypical trigger; even therapeutic doses cause hemolysis in G6PD-deficient persons), pamaquine, chloroquine (at high doses)
- Sulfonamides: Sulfamethoxazole, sulfanilamide, sulfacetamide (used in ophthalmologic preparations)
- Nitrofurantoin: Common cause of hemolysis; particularly problematic because it is frequently prescribed for urinary tract infections in primary care settings
- Dapsone: Used for leprosy and certain dermatologic conditions
- Rasburicase: Used for acute uric acid reduction in tumor lysis syndrome; contraindicated in G6PD deficiency
- Aspirin: At high doses (though therapeutic doses generally safe)
- Phenazopyridine: Used for dysuria
Class B Drugs—Moderate Risk (Hemolysis with High Doses or Specific Circumstances)
- NSAIDs: Ibuprofen, naproxen (particularly at high doses)
- Sulfonamide-containing antibiotics: Trimethoprim-sulfamethoxazole (TMP-SMX)
- Sulfonylureas: Some antidiabetic agents
- Acetaminophen: Usually safe at therapeutic doses
- Quinine: Antimalarial and antiarrhythmic
Fava Bean Ingestion
Fresh fava beans (Vicia faba) contain divicine and isouramil, potent oxidizing agents that trigger acute hemolytic crises in G6PD-deficient individuals—a phenomenon so characteristic that the disease is sometimes called "favism." The severity correlates with bean consumption quantity and G6PD enzyme activity level. Dried or cooked fava beans are safer due to oxidative degradation of the triggering compounds.
Infections as Oxidative Stressors
Both bacterial and viral infections can precipitate hemolytic episodes, independent of specific medications. Common triggers include urinary tract infections, pneumonia, and acute viral illness. Interestingly, infection may be the precipitating factor in patients taking medications theoretically at low hemolysis risk, suggesting infection-induced oxidative stress lowers the threshold for clinical hemolysis. Malaria parasites are particularly important in endemic populations and represent a biological example of how G6PD deficiency confers malaria-protective effects while carrying the risk of hemolysis.
Other Oxidative Stressors
- Hypoglycemia: Metabolic stress increases ROS production
- Acidosis: Cellular stress increases oxidative burden
- Surgical stress and anesthesia: Perioperative oxidative stress can trigger hemolysis
- Severe exercise: Rare but documented cause of hemolysis in susceptible individuals
- Newborn period: Jaundice and hemolysis are common presentations in neonates due to immature antioxidant systems and exposure to environmental oxidative stressors
Asymptomatic Presentation (Most Common)
The majority of individuals with G6PD deficiency remain asymptomatic throughout life, particularly those with Class III-IV variants or heterozygous females with favorable X-inactivation patterns. These individuals may be discovered incidentally on screening (family history, ethnic background, or routine blood count) or remain completely undiagnosed until exposure to an oxidative trigger. This highlights the importance of proactive case identification in at-risk populations.
Acute Hemolytic Crisis—Cardinal Presentation
Typically occurs 24-72 hours after exposure to an inciting oxidative trigger (medication, infection, or fava bean ingestion). Patients present with:
- Jaundice/icterus: Yellow discoloration of sclera and skin due to hyperbilirubinemia (indirect predominance). Jaundice may be dramatic and develop rapidly over hours, distinguishing it from other causes of hemolytic anemia.
- Dark urine: Tea-colored or cola-colored urine due to elevated urobilinogen and myoglobinuria in severe cases. The color persists or worsens despite increasing urine output, differentiating it from dilute urine.
- Hemoglobinuria: Passage of free hemoglobin in urine (positive dipstick without RBCs on microscopy—a classic dissociation that narrows differential diagnosis). This indicates sufficiently severe hemolysis to exceed renal haptoglobin binding capacity.
- Fatigue and malaise: Due to anemia and hemolytic crisis physiology
- Dyspnea and tachycardia: Compensatory responses to anemia; typically mild unless hemoglobin drops dramatically
- Abdominal discomfort: Splenic pain from splenic enlargement and infarction (rare), or visceral congestion
- Fever: May reflect underlying infection triggering the crisis or be part of the systemic inflammatory response to hemolysis
Physiological Basis of Symptoms
The symptom constellation reflects acute extravascular hemolysis with bilirubin production exceeding conjugation capacity (jaundice), RBC destruction releasing intracellular contents (hemoglobinuria, elevated indirect bilirubin), and compensatory reticulocytosis with associated metabolic demands (fatigue, tachycardia).
Neonatal Presentation
G6PD deficiency is a common cause of neonatal jaundice in at-risk populations, presenting as:
- Visible jaundice within first 24-48 hours of life (unusually early onset)
- Severe hyperbilirubinemia with risk of kernicterus
- Triggering factors include infection, acidosis, or hypoglycemia
- Hemolytic work-up reveals elevated indirect bilirubin with normal direct bilirubin, elevated reticulocyte count, and positive Coombs test (direct antiglobulin test) is notably negative (key distinguishing feature from other causes of hemolytic disease)
- Asian and Mediterranean variants carry higher risk of severe neonatal jaundice than African variants
Chronic Hemolytic Anemia (Class I Variants)
Rare patients with severe Class I G6PD variants (particularly Mediterranean and Asian types) experience chronic hemolysis even without oxidative triggers, resulting in:
- Baseline anemia (hemoglobin 7-10 g/dL)
- Persistent indirect hyperbilirubinemia and jaundice
- Splenomegaly (from chronic RBC destruction and extramedullary hematopoiesis)
- Risk of cholelithiasis from chronic bilirubin overproduction
- These patients may require chronic transfusion support in severe cases
Physical Examination Findings
- Jaundice: Scleral icterus and skin yellowing; severity correlates with indirect bilirubin level
- Pallor: Conjunctival and mucous membrane pallor reflecting anemia
- Splenomegaly: Palpable spleen due to increased hematopoiesis and RBC sequestration; massive splenomegaly is unusual and suggests chronic hemolysis or concurrent pathology
- Tachycardia: Compensatory response to anemia
- Tachypnea: If severe anemia or secondary lactic acidosis
- Hepatomegaly: May occur with severe hemolysis due to hepatic bilirubin conjugation burden or extramedullary hematopoiesis
Important Clinical Variants and Atypical Presentations
- Delayed hemolytic response: Some patients present with hemolysis 3-7 days after drug exposure, sometimes after the offending agent has been discontinued, possibly reflecting ongoing cellular oxidative damage and ROS accumulation
- Females with severe phenotype: Heterozygous females with unfavorable X-inactivation (skewed toward the mutant allele) can present with hemolysis severity approaching hemizygous males, sometimes surprising clinicians expecting milder disease
- Stress-related hemolysis: Athletes engaging in intense exercise or individuals experiencing severe psychological stress have documented hemolytic episodes despite no medication or fava bean exposure
- Subclinical hemolysis: Some individuals have laboratory evidence of chronic low-grade hemolysis (elevated reticulocyte count, mild indirect hyperbilirubinemia) without symptomatic hemolytic crises
Clinical History and Risk Stratification
Begin with targeted history focusing on ethnicity (highest prevalence in African, Mediterranean, and Southeast Asian populations), family history of hemolytic anemia or neonatal jaundice, specific medication exposures (particularly antimalarials, sulfonamides, nitrofurantoin), fava bean ingestion, and recent infections. Document temporal relationship between exposure and symptom onset. Ask about previous episodes of jaundice, dark urine, or anemia. Female patients should be questioned about X-linked inheritance patterns in family history (affected males, carrier females). The classic vignette for board exams: African male presenting with acute hemolysis after starting TMP-SMX for UTI or primaquine for presumed malaria.
Blood Smear Microscopy—Critical Diagnostic Clue
Examine peripheral blood smear during acute hemolytic episode or shortly thereafter. Key findings include:
- Bite cells (blister cells): Erythrocytes with a single or multiple "bites" removed from the RBC body where Heinz bodies were removed by splenic macrophages; highly suggestive of G6PD deficiency when present with hemolysis. Found in 5-50% of cells during acute hemolysis; absence does not exclude diagnosis.
- Spherocytes: May be present if hemolysis is brisk, though less prominent than in hereditary spherocytosis
- Polychromasia: Increased reticulocytes staining blue-purple (polychromatic) on Wright-Giemsa reflecting reticulocytosis
- Nucleated RBCs: Severe hemolysis may trigger release of immature RBCs from bone marrow
- Heinz bodies: Not visible on standard Wright-Giemsa but appear on supravital staining (crystal violet or brilliant cresyl blue staining). Multiple small densities within RBCs. This finding is highly specific
Immediate stabilisation
- Remove the oxidant: stopping the offending drug, fava beans, or treating the precipitating infection is the single most important intervention — hemolysis is self-limited once the oxidant is withdrawn because reticulocytes released in response carry near-normal G6PD activity.
- Isotonic IV fluids: maintain renal perfusion and urine flow to limit free hemoglobin/pigment cast deposition in tubules; monitor urine output, creatinine, and potassium (hemolysis releases intracellular K+).
- Serial monitoring: hemoglobin, reticulocytes, LDH, indirect bilirubin, and haptoglobin every 12–24 hours until the nadir passes (usually days 3–7).
First-line therapy
- Supportive care alone suffices for most episodes; there is no drug that restores G6PD activity.
- Packed RBC transfusion for symptomatic anemia, hemodynamic compromise, or a rapidly falling hemoglobin. AABB restrictive-transfusion guidance (a threshold near 7 g/dL in stable hospitalized adults) is a starting point, but ongoing brisk hemolysis justifies earlier transfusion.
- Neonates: intensive phototherapy, with exchange transfusion at the escalation thresholds in the AAP 2022 hyperbilirubinemia guideline; G6PD deficiency is explicitly listed there as a neurotoxicity risk factor that lowers treatment thresholds.
Escalation
- Renal replacement therapy for pigment-induced acute kidney injury with refractory hyperkalemia, acidosis, or volume overload.
- Exchange transfusion for massive intravascular hemolysis or neonatal bilirubin approaching exchange level.
- Folic acid supplementation in the rare chronic (Class I) nonspherocytic hemolytic anemia phenotype to support sustained erythropoiesis.
Definitive management
- Prevention is definitive: a documented allergy-style alert listing oxidant drugs and fava beans. Splenectomy is not indicated for ordinary episodic favism and is reserved, rarely, for transfusion-dependent Class I disease.
Contraindicated
- Rasburicase: FDA boxed warning — causes severe hemolysis and methemoglobinemia in G6PD deficiency; screen before use in tumor lysis syndrome.
- 8-aminoquinolines: FDA labeling for tafenoquine requires documented normal G6PD activity; CDC malaria guidance requires G6PD testing before primaquine radical cure.
- Methylene blue: ineffective and potentially hemolytic for methemoglobinemia in these patients (its action requires NADPH); use ascorbic acid or exchange transfusion.
- Dapsone, nitrofurantoin, sulfonamides, phenazopyridine in known deficiency.
Emergencies
- Acute severe hemolytic anemia with cardiovascular collapse: massive oxidant load overwhelms residual NADPH, producing hemoglobin fall over 24–48 hours; signalled by pallor, tachycardia, hypotension, and a precipitous hemoglobin drop with soaring LDH and undetectable haptoglobin. Requires urgent transfusion.
- Pigment (hemoglobinuric) acute kidney injury: free hemoglobin exceeds haptoglobin binding, filters into tubules, and causes cast obstruction, direct tubular oxidant injury, and vasoconstriction from nitric-oxide scavenging. Signalled by cola-colored urine, a dipstick positive for blood with no RBCs on microscopy, and rising creatinine. Most likely with the Mediterranean variant and favism.
- Acute bilirubin encephalopathy/kernicterus in neonates: unconjugated bilirubin generated faster than an immature UGT1A1 system can conjugate; signalled by lethargy, poor feeding, hypertonia, and retrocollis-opisthotonos. The AAP 2022 hyperbilirubinemia guideline treats G6PD deficiency as a neurotoxicity risk factor mandating lower treatment thresholds.
- Hyperkalemia and arrhythmia: intracellular potassium released from lysed erythrocytes, compounded by AKI; look for peaked T waves.
Subacute and chronic
- Aplastic crisis: superimposed parvovirus B19 infection halts erythroid precursors; signalled by falling hemoglobin with an inappropriately low reticulocyte count — the reticulocytopenia distinguishes it from a hemolytic crisis.
- Pigment (calcium bilirubinate) gallstones: chronic bilirubin overproduction; presents as biliary colic or cholecystitis, more common with coinherited Gilbert syndrome.
- Chronic nonspherocytic hemolytic anemia in Class I variants, with splenomegaly and growth impairment in children.
Treatment-related
- Transfusional iron overload in the rare chronically transfused patient; signalled by rising ferritin and transferrin saturation, with cardiac and hepatic deposition.
- Alloimmunization and delayed hemolytic transfusion reaction: a falling hemoglobin days after transfusion with a newly positive direct antiglobulin test — note the DAT is negative in G6PD hemolysis itself.
- Methemoglobinemia after rasburicase or oxidant exposure: cyanosis unresponsive to oxygen with a saturation gap; methylene blue is unsafe here.
- Post-splenectomy sepsis from encapsulated organisms in the rare surgical patient.
- The buzzword pair: bite cells on Wright-Giemsa and Heinz bodies on supravital stain (crystal violet, brilliant cresyl blue). Heinz bodies are invisible on the routine smear — if the stem says "routine stain showed no inclusions," that does not exclude the diagnosis.
- Single best next step in an acute crisis: stop the offending oxidant and give IV fluids. Do not order a G6PD enzyme assay first, and do not transfuse reflexively in a stable patient.
- The timing trap examiners love: a G6PD enzyme assay drawn during or immediately after hemolysis can be falsely normal, because the surviving and newly released reticulocytes are enzyme-rich. Repeat testing roughly 3 months after the episode (or after reticulocytosis resolves).
- The association tested most: X-linked recessive inheritance — an affected boy with an unaffected mother who has affected brothers. Remember that skewed lyonization can make a heterozygous female symptomatic, so "the patient is female" does not rule it out.
- Drug list worth memorizing: primaquine/tafenoquine, dapsone, nitrofurantoin, sulfonamides (TMP-SMX), phenazopyridine, and rasburicase (FDA boxed warning; screen before treating tumor lysis syndrome). Tafenoquine labeling and CDC malaria guidance require documented G6PD activity before use.
- Coombs is negative. A negative direct antiglobulin test with hemolysis, indirect hyperbilirubinemia, low haptoglobin, and high LDH points to an intrinsic RBC defect — the common distractor is autoimmune hemolytic anemia, which is DAT-positive.
- Distinguish from pyruvate kinase deficiency: PK deficiency is autosomal recessive, causes chronic hemolysis without an oxidant trigger, shows echinocytes rather than bite cells, and features high 2,3-BPG with a right-shifted oxygen curve.
- Neonatal jaundice appearing in the first 24–48 hours in an infant of Mediterranean or Asian ancestry: think G6PD. Per the AAP 2022 hyperbilirubinemia guideline, G6PD deficiency is a neurotoxicity risk factor that lowers phototherapy and exchange thresholds.
- Evolutionary hook: partial protection against Plasmodium falciparum explains the geographic distribution — the same balanced-polymorphism logic as sickle trait and thalassemia.