Cryptococcal meningitis should be suspected in immunocompromised patients with fever, headache, or any central nervous system symptoms or signs. Immunocompetent patients with subacute or chronic meningitis should also be evaluated for cryptococcal meningitis. A definite diagnosis can be established with CSF study. If the patient has focal neurological deficits, lumbar puncture (LP) should be performed only after imaging. The opening pressure is usually high, but when compared to HIV-positive patients, HIV-negative patients do not show much elevation. Cell count is increased, and mononuclear cells predominate in both HIV and non-HIV patients. Elevated protein and low sugar levels are other features. India ink stain shows encapsulated yeast, and fungal culture yields growth in 3–5 days. Additionally, cryptococcal antigen can be detected with latex agglutination assay, enzyme-linked immunosorbent assay, or lateral flow assay.

Case 1 — A 55 yr old female was admitted with a history of headache, irregular fever and vomiting of one-month duration followed by altered behaviour for a week. She was absolutely healthy prior to the onset of symptoms. There was no prior history of COVID-19 infection, and she was not vaccinated against COVID-19. General examination showed a confused patient (12 on the Glasgow Coma Scale) and central nervous system examination showed neck stiffness. All other systems were normal. LP was performed (Table 1). CSF culture had C. neoformans growth and liposomal amphotericin B was initiated. On day 11 of amphotericin administration, the patient developed papular eruptions over the chest and back with associated itching. Amphotericin was stopped, and fluconazole 800 mg tablet was started. Her CBC showed persistent lymphopenia (Table 1).

Chest radiography and ultrasonography findings were normal. In view of persistent lymphopenia, a COVID-19 antibody test was performed and was found positive. Lymphocyte subset analysis performed using flow cytometry showed absolute lymphocytopenia. The patient responded to fluconazole treatment and was discharged on fluconazole. On one-month follow-up, her CBC was returning to normal and her lymphocyte count was also improving.

Case 2 — A 47 yr old male who had a category A COVID-19 infection two months back, was admitted with a history of headache and vomiting for 1 month. On examination, he was conscious but disoriented. His pulse rate was 68 beats/min, blood pressure was 110/70 mm Hg, and saturation was 99% in room air. The systemic examinations were within normal limits, except for neck stiffness.

LP was performed and the opening pressure was low. Cell count showed lymphocytosis. Protein levels were high, while sugar levels were low (Table 2). India ink preparation showed cryptococci. CSF Gram staining showed gram-positive round budding yeast, and CSF cryptococcal antigen was also positive. CSF culture yielded cryptococci (Figure 1).

Figure 1

Table 2

Investigations	Day 1 admission	Day 3	Day 7	Day 20
Hemoglobin, g/dL	11	8.2	8.3	8.8
Total leukocyte count (per μL)	5800	4900	4000	4200
Differential Count (%)	N80,L11,M9	N82, L9,M9
Platelet(x105 per μL)	0.48	0.05	0.12	0.95
ESR (mm/hr)	14
Urea (mg/dL)	32	10	10	10
Creatinin(mg/dL)	0.5	0.5	0.5	0.8
Serum Sodium, mEq/L	130			134
Serum Potassium, mEq/L	3.4			3.9
LFT	Normal			Normal
Blood Glucose, mg/dL	99	110		128
Hb A1c ( %)	5.8
HIV / HBsAg / HCV ELISA	Negative
CSF Opening pressure Protein Sugar Cells India ink stain Fungal culture	Day 1 100 cm H2O 290mg/dL 8mg/dL NIL Fungal yeast seen Cryptococci grown	Day 14 110 cm H2O 275mg/dL 34mg/dL 117/µL L 80, N20 Few yeast cells seen No growth in culture
CD3 CD4 CD8 CD19 CD56	312 (Normal 570-2400/cu.mm) 34 (430-1800/cu.mm) 254 (210-120/cu.mm) 149 (91-610/cu.mm) 86 (78-470/cu.mm)

The patient was started on liposomal amphotericin B, but his cell count started to decline, hence, he was switched to fluconazole 800 mg. Peripheral smear examination showed thrombocytopenia and bone marrow examination showed focal megaloblastic changes, hence, vitamin B12 supplementation was initiated. The patient showed significant improvement. Since the patient had cryptococcal meningitis, he was evaluated for immunosuppression. A CD4+ cell count of 55 was noted. HIV antibody test and HIV RNA polymerase chain reaction were negative. Because CBC showed persisting lymphopenia one month after COVID-19 recovery, lymphocyte subset analysis was performed (Table 2). In view of profound CD4+ cell depletion, cotrimoxazole prophylaxis was initiated. The patient had hyponatraemia during hospitalisation, which improved with treatment. Magnetic resonance imaging showed a leptomeningeal enhancement and contrast-enhancing lesion over the left hemi-pons, suggestive of cryptococcoma. IgG Toxoplasma test results were negative. The patient showed a good response to the treatment.

Case 3 — A 49 yr old female was admitted with a history of headache of 2 weeks’ duration. She initially received analgesics from a local hospital. The patient had a category A COVID-19 infection, one month prior to the onset of headache, and was quarantined at home. Physical examination revealed an obese, delirious patient. Examination of skin showed scar marks of exaggerated insect bite reaction. CNS examination revealed right lateral rectus palsy and neck stiffness. LP was performed, and both opening pressure and cell count were high, protein level was elevated, and sugar was low, corresponding to blood sugar (Table 3). CSF gram staining revealed gram-positive yeasts, and India ink showed numerous cryptococci with negative staining capsules. The patient’s HIV test result was positive, and the CD4+ count was very low. The patient was initiated on intravenous amphotericin and flucytosine along with anti-oedema measures but succumbed to the illness on the 4th day of hospitalisation. There was no evidence of any other opportunistic infection.

Following the COVID-19 pandemic, the world of infectious diseases has changed into pre-COVID and post-COVID eras. The aetiology, pathophysiology, and clinical spectrum of various clinical syndromes has changed after the COVID-19 pandemic. Each syndrome complex should be approached with a broad spectrum of differential diagnoses and clinical investigations should be planned accordingly.

In this study, we describe three cases of cryptococcal meningitis which presented with fever, headache, and altered behaviour. On evaluation, only one patient had a classical predisposing condition, in this case HIV infection, which was diagnosed while evaluating meningitis. HIV alters Th1 Th2 balance with higher IL4 activity than IFNγ. Additionally, CD4+T cell granulysin-mediated killing of cryptococci does not occur in HIV infection. ^{6, 7} Even with a very low CD4+ count, the patient was asymptomatic. Whether preceding COVID-19 has any role in precipitating this opportunistic infection by rapidly depleting CD4 cells is yet to be determined. During this pandemic, we observed that Pneumocystis pneumonia also precipitated soon after COVID-19 infection in asymptomatic HIV-infected individuals. CD4+ count less than 50 is a high-risk factor for severe cryptococcal meningoencephalitis with high fatality. Other predictors of poor outcome include high CSF cryptococcal antigen level (titre> 1:1024 by latex agglutination assay, > 1:4000 by lateral flow assay), low body weight, poor CSF inflammatory response (< 20 cells/µL), and altered mental status on presentation. 10 An abnormal mental status indicates increased intracranial pressure.

The other two patients did not present with any described risk factors for cryptococcal meningitis. One of them had isolated CD4+ cytopenia and the other had pan-lymphocytopenia due to COVID-19 infection. To date, there have been no reports regarding the average duration of lymphopenia in post-COVID-19 patients. Hence, these patients were kept under follow-up. Patient with CD4+ cytopenia had a normal lymphocyte count after two months of follow-up.

Qin et al. demonstrated a dysregulated immune system in a cohort of 452 patients with COVID-19 infection. The major observation in the study was profound lymphopenia, and a reduction in CD4+ lymphocyte count was more evident in severe cases. However, no significant change in the number of CD8+ and B cells was noted. Moreover, they reported that the percentage of naïve helper T cells increased, while the number of memory helper T-cells, suppressor T-cells, and Treg cells decreased. The authors concluded that the reduction in the immune response resulted in excessive inflammation, a characteristic of severe COVID-19 infection. ¹¹ On the contrary, the patients described in the present study did not have severe COVID-19 infections and did not require hospitalisation. Hence, there is a possibility that even mild cases of COVID-19 can develop lymphopenia and serious opportunistic infections.

COVID-19 infection dysregulates the immune system, and the extent is still not fully understood. The associated therapy-induced suppression, such as steroid and tozulizumab, is highlighted more frequently. Common viral, bacterial, and fungal infections can occur due to the use of immunosuppressants in COVID-19 infections. However, all three patients in this series were not on immunosuppressants and two had only mild COVID-19 infection, while one was asymptomatic. Hence, we suggest that post-COVID-19 patients should be vigilantly monitored for opportunistic infections which are not usually considered in ordinary people.

Only very few cases of cryptococcal meningitis have been reported in the post-COVID-19 individuals. One report was of a patient with renal transplant with decompensated cirrhosis and COVID-19 in whom Cryptococcus was identified in the blood and ascitic fluid 12. Another report was on a patient with cryptococcemia in COVID-19 infection, who received tozulizumab and steroids at a high dose. ¹³ The patients described in the present study did not have an underlying immunosuppressed state, except for one patient who had HIV. Hebah et al. reported a case of severe cryptococcal meningitis that developed in a previously healthy patient one week after treatment of SARS-CoV-2 infection with dexamethasone. 14 Diana Alegre-González et al. also reported a case of disseminated cryptococcal infection following severe COVID-19 infection treated with immunosuppressants. ¹⁵

To maintain normal immunity in any person, a balanced CD4+/CD8+ ratio, and Th1, Th2, and Th17 responses are needed. Any alternation renders the person at risk for various infections, including opportunistic infections. COVID-19, by itself, is well known to produce a disequilibrium of immunity and hence is associated with increased vulnerability to numerous infections. Moreover, various drugs used for severe infections also result in immune suppression. This factor should be considered when following up on post-COVID-19 patients. In addition, when unusual infections occur in the normal population, even if a history of COVID-19 is not obtained, a post-COVID-19 state should be ruled out by performing an antibody test, if the individual is not vaccinated. Previous reports have highlighted the reversal of immune suppression following recovery from COVID-19 infection, but the patients described in the present study showed persistent lymphopenia. This factor highlights the significance of prolonged observation of post-COVID-19 individuals, and further studies are needed in this regard to determine the duration of persistent lymphopenia in post-COVID-19 infection.