Wednesday, 30 June 2021


Written by Alexia Rubio Peral | Jorge Gaitán Pitera | Jose Miguel Manchón Castilla

Figure 1. Image taken in ordinary light (oc x10, obj x40). In which structures compatible with yeasts are observed in CSF.

A 36-year-old male patient from Brazil who has lived in Spain for 3 months, comes to the emergency unit with a 3-month history of holocranial and throbbing headache, although with progression in intensity. The patient denies risky sexual contacts, drug use, eating spoiled food and contact with animals.

A lumbar puncture is performed, along with basic analysis that show anemia and increase in CRP (1.4 mg / dL).

In the CSF count 53 red blood cells / μL, 5 leukocytes / μL and structures compatible with yeast (Image 1) are observed. CSF biochemistry shows glucose of 22 mg / dL (NV: 50-80) and proteins of 44.0 mg / dL (NV: 15-40).
imagen2Figure 2. Images taken in ordinary light (oc x10, obj x100). LCR sample in pre-stained slide (Testsimplets®). In A image, the presence of yeast structures (in violet) is observed along with unstained red blood cells. In B imag, we can see in detail what appears to be an encapsulated yeast together with a red blood cell.

Given the lack of India ink to visualize the capsules, it was decided to perform a differential staining using pre-stained slides (Testsimplets®) (figure 2) and a Gram stain (figure 3).

In the differential staining, we can observe yeast structures stained with violet surrounded by a capsule. On the Gram stain, we observe Gram-positive structures that are consistent with yeast.

Doctor is informed of the suspicion of a Cryptococcus infection, pending confirmation by microbiology, and he is encouraged to make a request for HIV infectious serology. The study is completed by performing a culture of the sample, and a request for a multiple panel of molecular biology of meningitis by FilmArray®.

imagen3Figure 3: Images taken in ordinary light (oc x10, obj x100). Gram stain of CSF, showing Gram-positive yeast-like structures.
The results show a positive serology for HIV (with a CD4 lymphocyte count of 65 cells / μL, which gives us an idea of how advanced the infection is) and a positive FilmArray® for Cryptococcus neoformans / gattii. An India ink stain is performed on the CSF sample (figure 4) once it is available in our laboratory.

HIV infection is confirmed as HIV-1 by immunoblot techniques, with a viral load of 803,000 copies / mL.

imagen4Figure 4. Image taken in bright field microscopy (oc x10, obj x40). Negative staining of CSF with India ink revealing the striking capsule surrounding the yeast.

The yeast Cryptococcus neoformans identified through MALDI-TOF is isolated from the culture on Sabouraud agar, finally confirming the diagnosis ofcryptococcal meningoencephalitis.

The diagnosis of suspected cryptococcal meningitis is achieved during the analysis of CSF through the observation of yeast structures, since, as there is no previous diagnosis of HIV or other pathologies such as immunosuppression or diabetes this assumption does not fall within the differential diagnosis of the patient upon arrival at the emergency unit.

Early diagnosis and treatment can help reduce mortality from cryptococcal meningitis, a 15% of AIDS-related mortality worldwide.

Negative staining with India ink constitutes a presumptive diagnostic methodology for cryptococcosis, being one of the most used methods in the laboratory, although in our case we achieved a good visualization of the cryptococcal capsule using the slide pre-stained.


  1. Gary M Cox, John R Perfect. Epidemiology, clinical manifestations, and diagnosis of Cryptococcus neoformans meningoencephalitis in patients with HIV. UpToDate. 2020
  2. Cathy A Petti, Christopher R Polage M. Molecular diagnosis of central nervous system infections. pToDate. 2015
  3. Codina MG, Cuero M de, Echevarría JE, Vicente. Diagnóstico microbiológico de las infecciones del sistema nervioso central. Procedimientos en Microbiología Clínica. SEIMC. 2010.

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