Case reports
Patient 1 (P1) is an otherwise healthy 41-year-old man from Colombia (South America) who presented at the age of 34 years with progressive precordial pain with dysphagia, dry cough, moderate dyspnea, and a weight loss of 26 kg. After a convulsive episode, he was admitted to the emergency room and a physical examination revealed neck stiffness, a grade 2 systolic pulmonary murmur, and purple skin papules on his nose (Fig. 1a). Lumbar puncture revealed intracranial hypertension (ICH), and cerebrospinal fluid (CSF) abnormalities; India ink staining of the CSF and a serum cryptococcal antigen lateral flow assay (CrAg-LFA) were positive for Cryptococcus spp. (Table 1). Contrast-enhanced magnetic resonance imaging (CE-MRI) of the brain revealed multiple nodular lesions in the supratentorial areas and the basal ganglia that were consistent with cryptococcosis (Fig. 1b). Gram and India ink staining of sputum samples revealed the presence of encapsulated yeasts consistent with Cryptococcus spp. (Table 1). Contrast-enhanced computed tomography (CE-CT) of the chest revealed a peripheral nodule in the upper segment of the lower lobe of the left lung and a mediastinal mass (Fig. 1c). A biopsy of the mediastinal mass was performed by video-assisted thoracoscopic surgery (VATS) and the specimen was stained with India ink, revealing the presence of encapsulated yeasts consistent with Cryptococcus spp. (Fig. 1d). An echocardiogram revealed a mobile mass within the left atrium suggestive of mycotic endocarditis (not shown). Blood tests revealed leukocytosis with neutrophilia, and high C-reactive protein (CRP) levels. An HIV test was negative and flow cytometry analyses of lymphocyte subpopulations and serum immunoglobulins (Ig) were normal, with the exception of high IgE levels (Tables 1 and 2). An upper gastrointestinal (GI) tract endoscopy detected an esophageal ulcer positive for Cryptococcus spp., and cultures of CSF and the mediastinal mass on Sabouraud dextrose agar (SDA) grew C. neoformans (Table 2), confirming the diagnosis of disseminated cryptococcosis. The patient was treated with a six-week course of IV liposomal amphotericin B (LAmB) (250 mg/day), and 5 flucytosine (5-FC) (1 g qid), according to susceptibility test results, and esophageal and bronchial stents were implanted to close the fistula. However, serial lumbar punctures revealed persistent ICH after four weeks, leading to the insertion of a ventriculoperitoneal shunt (VPS). Finally, negative cultures of CSF, blood, and bronchial and esophageal tissues were obtained, and negative results were obtained for cytological analyses of bronchoalveolar lavage (BAL), ruling out a diagnosis of PAP. A clinical improvement was observed and the patient was discharged on oral FLC (800 mg/day), on which he remains, and is doing well.
Table 1
Blood and CSF laboratory parameters and microbiologic and pathology analyses of various tissues from the three patients at diagnosis
| Patient 1 | Patient 2 | Patient 3 |
Age at onset (years), Sex | 34, Male | 40, Female | 44, Female |
Hemogram |
Hemoglobin (g/dL) | 12.8 (ref. 13–17) | 13.6 (ref. 12–15) | 15.2 (ref. 12–15) |
Hematocrit (%) | 45.1 (ref. 40–52) | 41 (ref. 36–47) | 44.1 (ref. 36–47) |
WBC (ref. 4–10 x 109/L) | 13.7 | 9.2 | 15.12 |
Neutrophils (ref. 2–8 x 109/L) | 8,8 | 4.83 | 11.34 |
Lymphocytes (ref. 1–4 x 109/L) | 1.235 | 0.901 | 2.57 |
Monocytes (ref. 0.2–0.8 x 109/L) | 0.547 | 0.561 | 0.6 |
Eosinophils (ref. < 0.5 x 109/L) | 0.0 | 0.128 | 1.3 |
Platelets (ref. 150–450 x 109/L) | 428 | 328 | 506 |
Blood urea nitrogen (ref. 8–21 mg/dL) | 13.9 | 13 | 13.2 |
Creatinine (ref. 0.8–1.3 mg/dL) | 0.95 | 0.71 | 0.75 |
C-reactive protein (ref. 0.01–0.82 mg/dL) | 3.7 | 5 | 0.7 |
HIV serum antibodies (ELISA) | Negative | Negative | Negative (2) |
HIV serum viral load (HIV-1 RNA/mL) | Negative | Negative | Negative |
Cerebrospinal fluid (CSF) |
Total protein (ref. 5–45 mg/dL) | 81,7 | 242 | 68 |
Glucose (ref. 45–80 mg/dL) | 22 | 31 | 48 |
WBC (ref. 0–5 cells/µL) | 200 | 50 | 92 |
Opening pressure (ref. 10–25 cm H2O) | 47 | 20 | 21 |
ADA (15–60 mg/dL) | 13.2 | 24 | 3 |
Microbiologic and histologic tests |
Gram/India ink staining of selected samples | Positive for Cryptococcus spp. (sputum, CSF) | Positive | Positive |
Serum cryptococcal antigen test and titer | 1:512 (LFA*) | 1:256 | 1:128 |
Sabouraud dextrose agar cultures of selected samples and biopsies | All positive for C. neoformans (sputum, CSF, peripheral blood, BAL*, mediastinal mass, esophageal ulcer, papular skin lesions, soft tissues and bone) | Positive for C. neoformans (CSF and mediastinal mass) | Positive for C. gattii (CSF and mediastinal mass) |
Minimum inhibitory concentration (MIC) | FLC < 2 | FLC < 2 | FLC 16 VRC < 0.125 |
Pathology findings and silver methenamine and mucicarmine staining of selected tissues | Severe active chronic inflammation Silver methenamine and mucicarmine staining positive for blastoconidia (esophagus, skin and mediastinal mass) | Extensive coagulation necrosis, chronic inflammation Silver methenamine and mucicarmine staining positive for blastoconidia (mediastinal mass) | Histiocytic pneumonia, chronic inflammation Silver methenamine and mucicarmine staining positive for blastoconidia (lung mass) |
Other |
Aerobic cultures | Negative | Negative | Negative |
PCR mycobacteria/Mycobacterium cultures | Negative | Negative | Negative at diagnosis Positive for M. tuberculosis 5 years later |
* FLC: Fluconazole; VRC: Voriconazole. BAL: bronchoalveolar lavage. LFA: lateral flow assay. |
Values in bold are outside the normal range for age. |
Table 2
Flow cytometry of lymphocyte subsets in peripheral blood, serum immunoglobulins and specific antibodies
| Patient 1* | Patient 2 | Patient 3* | Ref. values for age** |
WBC (cells/µL) | 7645 | NR | 6270 | 5900 (4600–7100) |
Lymphocytes (%) | 20 | NR | 30.2 | 32 (28–39) |
Lymphocytes (cells/µL) | 1529 | NR | 1894 | 2300 (1200–4100) |
Lymphocyte subsets |
CD3+ (%) | 56 | NR | 71.7 | 67 (50–91) |
CD3+(cells/µL) | 856 | 876 (700–2100) | 1358 | 1500 (780–3000) |
CD3+/CD4+ (%) | 36.2 | NR | 44.3 | 42 (28–64) |
CD3+/CD4+ (cells/µL) | 553 | 613 (300–1400) | 839 | 1000 (500–2000) |
CD3+/CD8+ (%) | 18.7 | NR | 23.5 | 22 (12–40) |
CD3+/CD8+ (cells/µL) | 286 | 265 (200–900) | 445 | 500 (200–1200) |
CD3+/CD4+/CD8+ (%) | 0.4 | NR | 0.4 | 0.26 (0.075–0.94) |
CD3+/CD4+/CD8+ (cells/µL) | 4 | NR | 6 | 12 (2–60) |
CD4/CD8 ratio | 1.9 | 2.3 | 1.9 | 1.9 (1.0-3.6) |
CD19+ (%) | 13.4 | NR | 8.5 | 10 (4–28) |
CD19+ (cells/µL) | 205 | NR | 161 | 230 (64–820) |
CD3−CD16+/CD56+ (%) | 24.1 | NR | 16.5 | 15 (5–49) |
CD3-CD16+/CD56+ | 368 | NR | 312 | 340 (100–1200) |
CD45+/CD14+ (%) | 7.7 | NR | 5.7 | 3–8 |
CD45+/CD14+ /µL | 591 | NR | 359 | 100–8000 |
Serum immunoglobulins |
IgG (mg/dL) | 1467 (540–1822) | 1098 | ND | (814–2047) |
IgA (mg/dL) | 200 (63–484) | 88 | ND | (81–538) |
IgM (mg/dL) | 213 (22–240) | 154 | ND | (42–600) |
IgE (IU/mL) | 1150.8 (0-100) | ND | ND | |
HB IgG (mU/mL) | 0.08 (< 10) | ND | ND | |
Rubella IgG (mU/mL) | 187 (< 10) | ND | ND | |
* Flow cytometry of lymphocyte populations performed on peripheral blood lymphocytes (PBLs) stained with fluorochrome-labeled mAbs. Cells were collected on a FACS Canto II (BD Biosciences, San José, CA) and the data were analyzed with FlowJo v8.2 (TreeStar, Ashland, OR) by gating on CD45+ leukocytes. Reference values for lymphocyte subsets from Schatorjé EJH, et al. Scand. J Immunol. 2012 vol. 75 (4) pp. 436 − 44.**For patient 2, only results from the BD Tritest™ (BD Biosciences) were available and reference values are as indicated in the hospital records. ND: not done. NR: not reported. |
Patient 2 (P2) is a 46-year-old otherwise healthy woman from Colombia (South America) who presented at the age of 40 years old shoulder pain of four months’ duration radiating to the lower back, together with paresthesia of the lower abdomen and difficulty walking. Physical examination revealed a painful abdomen with bilateral positive Lasègue sign, sensory and motor deficit, a decrease in the muscle strength of both lower limbs, right Achillean reflex clonus, and bilateral positive Babinski sign. The patient suffered from permanent bilateral vision loss of unknown cause that had started four years previously, with lens opacification in the left eye and a corneal leukoma in the right eye. CE-MRI revealed extensive fluid collections in the right shoulder and lumbar spine, extending to the retroperitoneal, dural and posterior mediastinum, along with spinal cord compression and instability and bone destruction between T9 and T11 (Fig. 1e-f). P2 underwent intraoperative lavage and debridement with T9-T11 fixation, and the tissue samples excised were stained with silver methenamine and mucicarmine, indicating the presence of Cryptococcus spp.; C. neoformans grew in cultures of CSF and the mediastinal mass on SDA (Table 1). Brain CE-MRI showed hydrocephalus and acute meningitis (Fig. 1g), and CSF analysis after lumbar puncture revealed high total protein concentration, normal glucose concentration, high levels of leukocytes, and the CrAg assay was positive for Cryptococcus spp. (Table 1). Initial blood testing revealed a normal whole blood count (WBC), mild lymphopenia, and high CRP concentration; an HIV test was negative (Table 1). T-lymphocyte counts, CD4+/CD8+ T-cell ratio, and serum Ig levels were normal (Table 2). P2 was treated with IV deoxycholate AmB (DAmB) (42 mg/day) and oral 5-FC (1,500 mg qid) for 14 days, followed by oral FLC (800 mg/day) for 12 weeks, and was discharged on oral FLC (200 mg/day) plus analgesics. Seven months later, the patient was re-hospitalized due to severe back pain radiating to the left hypochondrium. The chest CT scan revealed a mediastinal mass and pleural effusion (Fig. 1h), and P2 underwent VATS for lung pleurectomy, decortication, and biopsy of the mediastinal mass. Silver methenamine and mucicarmine staining of the mediastinal mass revealed Cryptococcus spp. The patient was placed on IV DAmB (42 mg/day) for two weeks, but developed respiratory failure due to loculated pleural effusion of the right hemithorax with secondary lung collapse, requiring a new pleurectomy with decortication. Throughout P2’s disease, radiological and BAL studies have consistently ruled out pulmonary alveolar proteinosis (not shown). The patient remains free of signs and symptoms of infection and is not currently receiving antifungal agents; she is, however, permanently paraplegic.
Patient 3 (P3) is a 44-year-old previously healthy woman from Colombia who developed progressive whole-head headaches with photophobia, nausea, and vomiting in 2014. Physical examination was unremarkable except for neck stiffness with no neurologic focalization. Brain CE-MRI findings were consistent with meningitis (Fig. 1i), and lumbar puncture revealed a normal opening pressure and a CSF with a mild increase in protein levels, normal glucose concentration and a high level of leukocytes (Table 1). A non-contrast chest CT-scan revealed a right lung mass in contact with the pleura and a diffuse bilateral ground-glass pattern (Fig. 1j). VATS was performed to obtain samples of the lung mass and the affected pleura. Silver methenamine staining of the lung sample was consistent with the presence of Cryptococcus spp., and C. gattii grew in cultures of lung and pleura tissues on SDA, confirming the diagnosis of disseminated cryptococcosis (Table 1). Initial blood tests revealed moderate leukocytosis with neutrophilia and eosinophilia, thrombocytosis, and normal CRP levels; an HIV test was negative (Table 1). Flow cytometry analysis showed the proportions of the principal lymphocyte subpopulations to be within the range of normal values, and serum Ig levels were not evaluated (Table 2). The patient started a six-week course of treatment with IV DAmB (1 mg/kg/day) plus oral FLC (600 mg/day). However, antimicrobial susceptibility testing for FLC revealed a minimal inhibitory concentration (MIC) of 16 µg/mL (Table 1). P3 was therefore transferred onto suppressive therapy with voriconazole (VRC) at a dose of 200 mg/12 h for nine months, leading to the resolution of meningitis. A few months later, she developed a progressive dry cough, severe dyspnea and lost 7 kg in body weight. A new lumbar puncture and hemogram yielded values within the normal range. However, a CrAg assay on CSF was positive for Cryptococcus spp. High-resolution CT (HRCT) of the lungs revealed an increase in interstitial involvement, with a "crazy-paving” pattern (Fig. 1k). A right lung biopsy was therefore performed by VATS and histological analysis demonstrated the presence of abundant foamy histiocytes with myxoid material and small oval transparent structures consistent with blastoconidia (not shown). In addition, the alveolar spaces were occupied by amorphous eosinophilic material composed of histiocytes, cholesterol crystals, and a proteinaceous material positively stained with periodic acid-Schiff (PAS) staining, consistent with PAP (Fig. 1l). Ziehl-Neelsen (ZN) staining for mycobacteria was negative, and lung tissue cultures were negative for aerobic bacteria, fungi, and mycobacteria. The patient received suppressive therapy with oral posaconazole (POS; 300 mg/day) for three years and remained clinically stable. Five years later, she consulted again for a persistent dry cough and weight loss. Chest HRCT showed a progression of pulmonary damage, with ground-glass and bilateral multilobar consolidations, solid nodules of up to 8 mm in diameter, and centrilobular nodules with a “budding tree” morphology. BAL and lumbar puncture were normal and cultures were negative. VATS was performed on the right lower lobe, and histological analysis of the specimen revealed the presence of multiple giant epithelioid cells with caseous necrosis. Methenamine silver and ZN staining was negative, but PCR (GeneXpert, Cepheid®) and cultures for mycobacteria were positive for multisusceptible Mycobacterium tuberculosis. P3 received a directly observed treatment short (DOTS) regimen with rifampicin (R), isoniazid (H), pyrazinamide (Z), and ethambutol (E) for two months, followed by four months of R-H treatment with clinical improvement, but with dyspnea on exertion. P3 has not undergone whole-lung lavage or required oxygen supplementation and she remains stable.
The etiological nature and severity of the cryptococcal infections in these three previously healthy adult patients prompted us to evaluate the possibility that neutralizing anti-GM-CSF auto-Abs might underlie the infectious diseases observed in these patients. We performed an ELISA for the detection of these auto-Abs in plasma samples from these three patients. As a control, we used plasma from a patient with PAP and high titers of neutralizing anti-GM-CSF auto-Abs (positive control). We also included plasma from a patient with high titers of anti-IFN-γ neutralizing auto-Abs, an APS-1 patient with high titers of anti-IL-17A, IL-17F, and IL-22 auto-Abs, and two healthy individuals. All three patients had high levels of anti-GM-CSF auto-Abs, in the range of the positive control, whereas none of the heathy individuals, the APS-1 patient, or the patient with auto-Abs against IFN-γ had auto-Abs against GM-CSF (Fig. 2a). We detected no neutralizing auto-Abs against type I IFNs in the three patients in the testing conditions used (Supplementary Fig. 1). Finally, we assessed the neutralizing activity of the plasma samples from the three patients by stimulating PBMCs from healthy individuals ex vivo with rhGM-CSF or IL-3 as a positive control to induce GM-CSF receptor-mediated or IL-3R-mediated phosphorylation of STAT5 (pSTAT5) in the presence of 10% plasma from healthy individuals, or of the patients, and then performing flow cytometry (23). Unlike control plasma from healthy individuals, plasma with high anti-GM-CSF auto-Ab titers from the three patients prevented GM-CSF-induced STAT5 phosphorylation in PBMCs from healthy individuals, whereas the level of IL-3-induced STAT5 phosphorylation was similar in cells incubated with control or patient plasma (Fig. 2b, Supplementary Fig. 2). These results strongly suggest that the presence of circulating neutralizing auto-Abs against GM-CSF was responsible for the disseminated cryptococcosis of these patients.