India Ink

India ink (or nigrosin) preparation is a rapid microscopic examination of CSF for encapsulated yeasts, mainly Cryptococcus neoformans and Cryptococcus gattii. A drop of centrifuged CSF sediment is mixed with a drop of India ink on a slide, covered with a coverslip, and examined at 100 and 400 times magnification.

The ink particles are excluded by the polysaccharide capsule, producing a clear halo around round, budding yeast cells 4 to 12 micrometres across, with refractile cell walls. The test takes 5 to 10 minutes and needs only a microscope, but it is largely replaced by the cryptococcal antigen lateral flow assay where available.

India ink has sensitivity of 70 to 90% in HIV-associated cryptococcal meningitis (burden is high, often over 1000 colony forming units/mL) and 30 to 50% in HIV-negative patients with lower fungal burden. Specificity is high (over 95%) with trained readers, but artefacts mimic yeasts. It remains useful in low resource settings that lack antigen tests and for rapid confirmation while culture and CrAg results are awaited.

Indications. Meningitis in an immunocompromised patient, especially HIV with CD4 under 100 cells/microlitre. Headache, fever and confusion with lymphocytic CSF and low glucose. Subacute or chronic meningitis. Raised intracranial pressure of unknown cause. Suspected cryptococcosis in any body fluid (urine, bronchoalveolar lavage, aspirates) in settings without antigen testing. Not for monitoring.

Specimen. CSF 1 to 2 mL, centrifuged at 3000 rpm for 10 minutes, with a drop of sediment used. Collected by lumbar puncture in a sterile tube. Examined without delay, as cryptococci remain viable at room temperature (do not refrigerate CSF kept for culture). Other fluids (bronchoalveolar lavage, urine, pus, tissue impressions) can be examined.

Reference values. Negative: no encapsulated yeast cells seen on the whole coverslip. Positive: round or oval budding yeasts with clear capsule halo against the dark background. Results can be graded by number of cells seen (few, moderate, many) as a rough indicator of burden.

Interpretation.

• Positive India ink with compatible clinical findings: cryptococcal meningitis is confirmed, and treatment starts urgently without waiting for culture. Opening pressure measurement and therapeutic drainage follow.
• Positive India ink and high yeast count correlates with high CSF cryptococcal antigen titre and raised opening pressure, and worse outcomes.
• Negative India ink does not exclude cryptococcal meningitis (up to 30% of HIV cases and 50 to 70% of non-HIV cases are negative). CrAg and culture are needed.
• Budding encapsulated yeasts in other fluids (BAL, pleural fluid, urine, blood) indicate disseminated or localised cryptococcosis.
• Capsule deficient strains, small capsule C. gattii and non-capsulated Candida or Histoplasma may be missed or misread.
• Results are reported in minutes, and the laboratory phones positive results.

Critical values and findings needing immediate action.

• Positive India ink on CSF: telephone the clinician immediately, start amphotericin based induction therapy, measure opening pressure and perform therapeutic lumbar puncture if raised (over 25 cmH2O).
• Positive India ink in a patient with raised pressure symptoms (severe headache, vomiting, visual loss, papilloedema): urgent drainage.
• Positive India ink with CD4 under 100 and not on antiretroviral therapy: plan delayed antiretroviral therapy start (4 to 6 weeks after antifungal therapy).
• Positive India ink in a patient on immunosuppressive drugs: reduce immunosuppression where possible and start treatment.

Interferences and pitfalls.

• Artefacts that can be mistaken for cryptococci: lymphocytes and other cells, air bubbles, fat globules, talc and starch particles, ink aggregates, red cells and other yeasts. Cryptococci have a typical thick capsule, narrow necked budding and refractile cell wall.
• Sensitivity depends on fungal burden, centrifugation of the sample, reader experience and number of fields examined. A thin film is better than a thick one.
• The test cannot differentiate C. neoformans from C. gattii, or confirm viability, and culture is required for species identification and susceptibility testing.
• A negative India ink is not reassuring in HIV-negative patients, and cryptococcal antigen is far more sensitive (over 99% in HIV).
• India ink of urine or blood is insensitive and not used for diagnosis.
• Contaminated ink bottles and prepared stock can introduce yeasts and bacteria, and fresh ink and control preparations are needed.
• Differential diagnosis of lymphocytic meningitis with low glucose includes tuberculosis, other fungi (histoplasmosis, coccidioidomycosis), neurosyphilis, listeriosis, carcinomatous meningitis and sarcoidosis.
• India ink does not replace opening pressure measurement, since raised pressure is the main cause of early morbidity.

Special groups.

• Children: rarely positive, but done in HIV-positive children with meningitis.
• Pregnancy: management with amphotericin B (flucytosine and fluconazole are teratogenic).
• Older adults: confusion may be the only sign.
• Non-HIV immunocompromised patients and immunocompetent patients with C. gattii: lower yeast load and negative smears more often, so antigen and culture are essential.
• Regions: in resource limited settings with high HIV prevalence, India ink combined with clinical assessment has directed life saving treatment, but it is being replaced by point-of-care CrAg assays, which are more sensitive and equally cheap.

Follow up tests. Cryptococcal antigen (CSF and serum), CSF fungal culture, CSF opening pressure, cell count, protein and glucose, repeat lumbar punctures (day 14 culture to document sterility). CD4 count and HIV viral load, creatinine and electrolytes during amphotericin. MRI or CT of the brain, ophthalmology, chest imaging, and susceptibility testing of the isolate.