Neuroimaging of Cryptococcal Meningitis in Patients without Human Immunodeficiency Virus: Data from a Multi-Center Cohort Study.

BACKGROUND: A clearer understanding is needed about the use of brain MRI in non-HIV patients with cryptococcal meningitis. METHODS: Cerebral CT and MRI were studied in 62 patients in a multicenter study of cryptococcal meningitis in non-HIV patients. CT was performed in 51 and MRI in 44. MRI results are reported for the images read at NIH for 29 of the 44 patients. CT reports obtained from the original REDCap database were added to calculate the incidence of normal findings. RESULTS: CTs were read as normal in 24 of 51 (47%), MRIs were normal in 10% (three of 29). The most characteristic lesions of cryptococcal meningitis on MRI were small basal ganglia lesions representing dilated perivascular spaces in 24% and basal ganglia lesions with restricted diffusion (infarcts) in 38%. In the 18 patients who received contrast, contrast-enhancing lesions, likely representing masses of cryptococci and inflammatory cells, were found in the basal ganglia in 22% and elsewhere in the brain in 22%. Meningeal enhancement was seen in 56%, ependymal enhancement in 24%, and choroid plexus enhancement in 11%. Hydrocephalus was found in five (18%), though increased intacranial pressure was not detected. Suboptimal imaging (n = 6), lack of contrast administration (n = 11) and lack of follow-up, however, markedly limited the accurate assessment of abnormalities in multiple cases. CONCLUSION: MRI characteristics of non-HIV cryptococcal meningitis include hydrocephalus, meningeal and ependymal enhancement and basal ganglia lesions. Optimal imaging is, however, necessary to maximize the diagnostic and prognostic usefulness of MRI.

Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, ß-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1ß in response to ß-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1ß and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1ß-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

Neutralizing GM-CSF autoantibodies in pulmonary alveolar proteinosis, cryptococcal meningitis and severe nocardiosis.

BACKGROUND: Anti GM-CSF autoantibodies (aAb) have been related to acquired pulmonary alveolar proteinosis (PAP) and described in cases of severe infections such as cryptococcosis and nocardiosis in previously healthy subjects. Whether there are different anti-GM-CSF autoantibodies corresponding to these phenotypes is unclear. Therefore, we examined anti-GM-CSF autoantibodies to determine whether amount or neutralizing activity could distinguish between groups. METHODS: Plasma samples gathered in the National Institute of Health from patients with anti GM-CSF aAb and either PAP (n = 15), cryptococcal meningitis (n = 15), severe nocardiosis (n = 5) or overlapping phenotypes (n = 6) were compared. The relative amount of aAb was assessed using a particle-based approach, reported as a mouse monoclonal anti-human GM-CSF as standard curve and expressed in an arbitrary Mouse Monoclonal Antibody Unit (MMAU). The neutralizing activity of the plasma was assessed by inhibition of GM-CSF-induced intracellular phospho-STAT5 (pSTAT5) in monocytes. RESULTS: Anti-GM-CSF aAb relative amounts were higher in PAP patients compared to those with cryptococcosis (mean 495 ± 464 MMAU vs 197 ± 159 MMAU, p = 0.02); there was no difference with patients with nocardiosis (430 ± 493 MMAU) nor between the two types of infections. The dilution of plasma resulting in 50% inhibition of GM-CSF-induced pSTAT5 (approximate IC50) did not vary appreciably across groups of patients (1.6 ± 3.1%, 3.9 ± 6% and 1.8 ± 2.2% in PAP patients, cryptococcosis and nocardiosis patients, respectively). Nor was the concentration of GM-CSF necessary to induce 50% of maximal GM-CSF-induced pSTAT5 in the presence of 10 MMAU of anti-GM-CSF aAb (EC50). When studying longitudinal samples from patients with PAP or disseminated nocardiosis, the neutralizing effect of anti-GM-CSF aAb was relatively constant over time despite targeted treatments and variations in aAb levels. CONCLUSIONS: Despite different clinical manifestations, anti-GM-CSF antibodies were similar across PAP, cryptococcosis and nocardiosis. Underlying host genetics and functional analyses may help further differentiate the biology of these conditions.

A Target Site Mutation Associated With Diamide Insecticide Resistance in the Diamondback Moth Plutella xylostella (Lepidoptera: Plutellidae) is Widespread in South Georgia and Florida Populations.

Laboratory colonies of diamondback moth (DBM) larvae were established from larvae collected from four sites in Georgia and Florida where diamide, specifically chlorantraniliprole, insecticide resistance was recently documented. Based on dose-response experiments, these colonies exhibited 109- to 4,298-fold resistance to chlorantraniliprole, compared to a commercially available susceptible control colony. Colonies exhibited 50- to 107-fold resistance to another diamide, cyantraniliprole, based on similar dose-response experiments. All colonies were screened for the presence of four known mutations in the ryanodine receptor (RyR), the target of diamide insecticides, previously associated with resistance in Asian DBM populations. One mutation, G4946E, was identified in colonies from all four field sites, but not the susceptible control colony. Three additional RyR target site mutations, E1338D, Q4594L, and I4790M, were not identified in any of the screened samples. The estimated allele frequency of the G4946E mutation in these colonies ranged from 32 to 90%. These data are consistent with recently reported chlorantraniliprole control failures in Georgia and Florida. It is likely that the G4946E mutation is currently an important contributing factor to chlorantraniliprole resistance in Georgia and Florida DBM populations.

Cryptococcus gattii Species Complex as an Opportunistic Pathogen: Underlying Medical Conditions Associated with the Infection.

The Cryptococcus gattii species complex has often been referred to as a primary pathogen due to its high infection frequency among apparently immunocompetent patients. In order to scrutinize the immune status of patients and the lineages of etiologic agents, we analyzed patient histories and the molecular types of etiologic agents from 135 global C. gattii cases. Eighty-six of 135 patients had been diagnosed as immunocompetent, although some of them had underlying medical issues, and 49 were diagnosed as immunocompromised with risk factors similar to those seen in Cryptococcus neoformans infection. We focused on the 86 apparently immunocompetent patients and were able to obtain plasma from 32 (37%) to analyze for the presence of autoantibodies against the granulocyte-macrophage colony-stimulating factor (GM-CSF) since these antibodies have been reported as a hidden risk factor for C. gattii infection. Among the 32 patients, 25 were free from any known other health issues, and 7 had various medical conditions at the time of diagnosis for cryptococcosis. Importantly, plasma from 19 (76%) of 25 patients with no recognized underlying medical condition showed the presence of GM-CSF autoantibodies, supporting this antibody as a major hidden risk factor for C. gattii infection. These data indicate that seemingly immunocompetent people with C. gattii infection warrant detailed evaluation for unrecognized immunologic risks. There was no relationship between molecular type and underlying conditions of patients. Frequency of each molecular type was related to its geographic origin exemplified by the overrepresentation of VGIV in HIV-positive (HIV+) patients due to its prevalence in Africa. IMPORTANCE The C. neoformans and C. gattii species complex causes cryptococcosis. The C. neoformans species complex is known as an opportunistic pathogen since it primarily infects immunocompromised patients. C. gattii species complex has been referred to as a primary pathogen due to its high infection frequency in apparently immunocompetent people. We analyzed 135 global cases of C. gattii infection with documented patient history. Eighty-six of 135 patients were originally diagnosed as immunocompetent and 49 as immunosuppressed with similar underlying conditions reported for C. neoformans infection. A significant number of C. gattii patients without known underlying conditions possessed autoantibodies against granulocytes-macrophage colony-stimulating factor (GM-CSF) in their plasma, supporting the presence of GM-CSF antibodies as a hidden risk factor for C. gattii infection. No relationship was found between C. gattii lineages and the underlying conditions except for overrepresentation of the molecular type VGIV among HIV+ patients due to the prevalence of VGIV in Africa.

Moderate levels of 5-fluorocytosine cause the emergence of high frequency resistance in cryptococci.

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.

Cutaneous hyalohyphomycosis due to Petriella setifera following traumatic inoculation in an immunocompetent host.

A common cause of subcutaneous mycoses in immunocompetent patients is traumatic inoculation. This is the first reported case of cutaneous infection with Petriella setifera in a human host. A 49-year-old immunocompetent man sustained a splinter from his wooden deck, which resulted in a chronic cutaneous lesion. Originally identified as Scedosporium species on microscopic examination, genome sequencing of the internal transcribed spaces (ITS) region of ribosomal DNA (rDNA) identified the mold as Petriella setifera, a common environmental fungus responsible for wood rot. The patient underwent excision and antifungal therapy with cure.

Outcomes in Previously Healthy Cryptococcal Meningoencephalitis Patients Treated With Pulse Taper Corticosteroids for Post-infectious Inflammatory Syndrome.

BACKGROUND: Cryptococcal meningoencephalitis (CM) is a major cause of mortality in immunosuppressed patients and previously healthy individuals. In the latter, a post-infectious inflammatory response syndrome (PIIRS) is associated with poor clinical response despite antifungal therapy and negative cerebrospinal fluid (CSF) cultures. Data on effective treatment are limited. METHODS: Between March 2015 and March 2020, 15 consecutive previously healthy patients with CM and PIIRS were treated with adjunctive pulse corticosteroid taper therapy (PCT) consisting of intravenous methylprednisolone 1 gm daily for 1 week followed by oral prednisone 1 mg/kg/day, tapered based on clinical and radiological response plus oral fluconazole. Montreal cognitive assessments (MOCA), Karnofsky performance scores, magnetic resonance imaging (MRI) brain scanning, ophthalmic and audiologic exams, and CSF parameters including cellular and soluble immune responses were compared at PIIRS diagnosis and after methylprednisolone completion. RESULTS: The median time from antifungal treatment to steroid initiation was 6 weeks. The most common symptoms at PIIRS diagnosis were altered mental status and vision changes. All patients demonstrated significant improvements in MOCA and Karnofsky scores at 1 month (P < .0003), which was accompanied by improvements in CSF glucose, white blood cell (WBC) count, protein, cellular and soluble inflammatory markers 1 week after receiving corticosteroids (CS) (P < .003). All patients with papilledema and visual field deficits also exhibited improvement (P < .0005). Five out of 7 patients who underwent audiological testing demonstrated hearing improvement. Brain MRI showed significant improvement of radiological findings (P = .001). CSF cultures remained negative. CONCLUSIONS: PCT in this small cohort of PIIRS was associated with improvements in CM-related complications with minimal toxicity in the acute setting.

Population diversity and virulence characteristics of Cryptococcus neoformans/C. gattii species complexes isolated during the pre-HIV-pandemic era.

Cryptococcosis has become a major global health problem since the advent of the HIV pandemic in 1980s. Although its molecular epidemiology is well-defined, using isolates recovered since then, no pre-HIV-pandemic era epidemiological data exist. We conducted a molecular epidemiological study using 228 isolates of the C. neoformans/C. gattii species complexes isolated before 1975. Genotypes were determined by URA5 restriction fragment length polymorphism analysis and multi-locus sequence typing. Population genetics were defined by nucleotide diversity measurements, neutrality tests, and recombination analysis. Growth at 37°C, melanin synthesis, capsule production, and urease activity as virulence factors were quantified. The pre-HIV-pandemic isolates consisted of 186 (81.5%) clinical, 35 (15.4%) environmental, and 7 (3.1%) veterinary isolates. Of those, 204 (89.5%) belonged to C. neoformans VNI (64.0%), VNII (14.9%) and VNIV (10.5%) while 24 (10.5%) belonged to C. gattii VGIII (7.5%), VGI (2.6%) and VGII (0.5%). Among the 47 sequence types (STs) identified, one of VNII and 8 of VNIV were novel. ST5/VNI (23.0%) in C. neoformans and ST75/VGIII (25.0%) in C. gattii were the most common STs in both species complexes. Among C. neoformans, VNIV had the highest genetic diversity (Hd = 0.926) and the minimum recombination events (Rm = 10), and clinical isolates had less genetic diversity (Hd = 0.866) than environmental (Hd = 0.889) and veterinary isolates (Hd = 0.900). Among C. gattii, VGI had a higher nucleotide diversity (π = 0.01436) than in VGIII (π = 0.00328). The high-virulence genotypes (ST5/VNI and VGIIIa/serotype B) did not produce higher virulence factors levels than other genotypes. Overall, high genetic variability and recombination rates were found for the pre-HIV-pandemic era among strains of the C. neoformans/C. gattii species complexes. Whole genome analysis and in vivo virulence studies would clarify the evolution of the genetic diversity and/or virulence of isolates of the C. neoformans/C. gattii species complexes during the pre- and post-HIV-pandemic eras.

Cryptococcus neoformans/gattii Species Complexes from Pre-HIV Pandemic Era Contain Unusually High Rate of Non-Wild-Type Isolates for Amphotericin B.

INTRODUCTION: The Cryptococcus neoformans/gattii species complexes are a leading cause of fatality among HIV-infected patients. Despite the unavailability of clinical breakpoints (CBPs) for antifungal agents, epidemiological cutoff values (ECVs) were recently proposed, and non-wild-type isolates for polyenes and azoles are being increasingly reported. However, the distributions of the susceptibility patterns for pre-HIV-era isolates have not been studied. METHODS: We determined the in vitro antifungal susceptibility patterns of 233 Cryptococcus isolates, collected at the National Institutes of Health, USA, in pre-HIV pandemic era, to study minimum inhibitory concentrations (MICs) to the important drugs for cryptococcosis and to compare the results with strain genotypes. Amphotericin B susceptibility was compared to published ECV of C. neoformans. RESULTS: The 233 Cryptococcus strains consisted of 89.7% C. neoformans species complex and 10.3% C. gattii species complex. Most were from clinical sources (189, 81.1%), and the major molecular type was VNI (146, 62.7%). The highest geometric mean (GM) was observed for fluconazole (GM = 0.96 µg/mL) while the lowest was for itraconazole (GM = 0.10 µg/mL). MICs to fluconazole in C. gattii species complex were significantly higher than C. neoformans species complex (p < 0.001). Moreover, C. neoformans/VNI strains showed significantly higher MICs than others such as C. neoformans/VNII to fluconazole (p < 0.0001) and C. deneoformans/VNIV to amphotericin B (p = 0.022) and fluconazole (p = 0.008). In our collection of 167 clinical C. neoformans species complex strains, 85 (50.9%), 24 (14.4%), and 3 (1.8%) strains had an amphotericin B (AMB)-MIC of 1, 2, and 4 µg/mL, respectively. The high percentage (66.9%, 79/118 strains) of non-wild-type clinical C. neoformans VNI strains, using an AMB-ECV of 0.5 µg/mL, was found. Moreover, 25 of 28 (89.3%) C. neoformans VNI strains from environmental and veterinary sources also had AMB-MICs above 0.5 µg/mL. In general, there was no significant difference in GM AMB-MIC of the clinical strains isolated from patients with (35 patients) and without (78 patients) prior AMB treatment (0.85 vs 0.76; p = 0.624). GM MIC of the environmental strains was not significantly different from that of the prior AMB-treatment strains (0.98 vs 0.76, p = 0.159) and the post-AMB-treatment strains (0.98 vs 0.85, p = 0.488). CONCLUSION: The high rate of non-wild-type among these otherwise naive isolates to amphotericin B is unexpected. Confirmation with more strains from a later era is needed.

HIV-Negative Cryptococcal Meningoencephalitis Results in a Persistent Frontal-Subcortical Syndrome.

Twenty-seven previously healthy (of 36 consecutive eligible patients), HIV-negative cryptococcal meningoencephalitis (CM) patients underwent comprehensive neuropsychological evaluation during the late post-treatment period (1.3-4 years post diagnosis), assessing attention, language, learning, memory, visuospatial, executive function, information processing, psychomotor functioning, as well as mood symptoms. Seven of eight domains (all except attention) showed increased percentages of CM patients scoring in the less than 16th percentile range compared to standardized normative test averages, adjusted for education level and age. Comparison with a matched archival dataset of mild cognitive impairment/Alzheimer's disease patients showed that CM patients exhibited relative deficits in psychomotor and executive function with fewer deficits in memory and learning, consistent with a frontal-subcortical syndrome. MRI evaluation at the time of testing demonstrated an association of lower neuropsychological functioning with ventriculomegaly. These studies suggest that CM should be included in the list of treatable causes of dementia in neurological work ups. Future studies are needed to identify diagnostic and treatment regimens that may enhance neurological function after therapy.

Audiologic and Otologic Complications of Cryptococcal Meningoencephalitis in Non-HIV Previously Healthy Patients.

OBJECTIVE: To identify audiologic and otologic outcomes in previously healthy non-HIV patients with cryptococcal meningoencephalitis (CM). STUDY DESIGN: Retrospective case review of a subset of patients recruited in a prospective observational study following previously healthy individuals who developed CM. SETTING: Tertiary referral center, National Institutes of Health Clinical Center. PATIENTS: Previously healthy adult patients with CM without immune suppressive therapy before disease onset. INTERVENTIONS: Diagnostic evaluations included audiometry, acoustic immittance, otoacoustic emissions, and auditory brainstem response studies, in addition to neurotologic assessment. RESULTS: Twenty-nine patients (58 years) underwent audiologic evaluation between 6 months and 3.5 years after CM diagnosis; 21 patients were seen for longitudinal assessment with an average duration of follow up of 20.3 months. Nearly three-quarters (73%) of the cohort presented with hearing loss, most commonly (90%) sensorineural in origin. The most frequent degree of loss was mild and then moderate, although some patients had severe or profound impairment. Hearing loss improved (43%) or remained stable (38%) in most cases. Ears with internal auditory canal enhancement on magnetic resonance imaging (MRI) had significantly more hearing loss than those without enhancement, although a similar finding was not observed with gyral enhancement or the presence of ependymitis or ventricular volume expansion. Hearing loss was not associated with reduced cerebrospinal fluid (CSF) glucose, CSF total protein, cryptococcal antigen, or total cell count. CONCLUSIONS: Hearing loss is a common manifestation of cryptococcal meningitis in previously healthy patients and may involve a cochlear or neural site of lesion, or both. Routine surveillance of hearing in patients is recommended, regardless of symptomatology, to ensure early and appropriate intervention and care.

Protracted course of disseminated adenovirus disease with necrotizing granulomas in the liver.

A 52- year-old male with chronic lymphocytic leukemia was hospitalized with disseminated adenovirus disease. More than a month following recovery, hepatic necrotizing granulomas secondary to adenovirus were found. This case illustrates the protracted course that adenovirus disease may take and emphasizes an unusual presentation with hepatic necrotizing granulomas.

Management Practices for Methicillin-Resistant Staphylococcus aureus Bacteremia by Adult Infectious Diseases Physicians.

To assess current use of vancomycin for methicillin-resistant Staphylococcus aureus bacteremia, we surveyed adult infectious disease physicians. Most respondents reported personal experience with infections failing to respond to vancomycin despite minimum inhibitory concentration data indicating susceptibility. In a hypothetical case of such an infection, most would change to daptomycin with or without other agents.

Sterol uptake and sterol biosynthesis act coordinately to mediate antifungal resistance in Candida glabrata under azole and hypoxic stress.

Pathogenic fungi, including Candida glabrata, develop strategies to grow and survive both in vitro and in vivo under azole stress. However, the mechanisms by which yeast cells counteract the inhibitory effects of azoles are not completely understood. In the current study, it was demonstrated that the expression of the ergosterol biosynthetic genes ERG2, ERG3, ERG4, ERG10, and ERG11 was significantly upregulated in C. glabrata following fluconazole treatment. Inhibiting ergosterol biosynthesis using fluconazole also increased the expression of the sterol influx transporter AUS1 and the sterol metabolism regulators SUT1 and UPC2 in fungal cells. The microarray study quantified 35 genes with elevated mRNA levels, including AUS1, TIR3, UPC2, and 8 ERG genes, in a C. glabrata mutant strain lacking ERG1, indicating that sterol importing activity is increased to compensate for defective sterol biosynthesis in cells. Bioinformatic analyses further revealed that those differentially expressed genes were involved in multiple cellular processes and biological functions, such as sterol biosynthesis, lipid localization, and sterol transport. Finally, to assess whether sterol uptake affects yeast susceptibility to azoles, we generated a C. glabrata aus1∆ mutant strain. It was shown that loss of Aus1p in C. glabrata sensitized the pathogen to azoles and enhanced the efficacy of drug exposure under low oxygen tension. In contrast, the presence of exogenous cholesterol or ergosterol in medium rendered the C. glabrata AUS1 wild­type strain highly resistant to fluconazole and voriconazole, suggesting that the sterol importing mechanism is augmented when ergosterol biosynthesis is suppressed in the cell, thus allowing C. glabrata to survive under azole pressure. On the basis of these results, it was concluded that sterol uptake and sterol biosynthesis may act coordinately and collaboratively to sustain growth and to mediate antifungal resistance in C. glabrata through dynamic gene expression in response to azole stress and environmental challenges.

Choroid Plexitis and Ependymitis by Magnetic Resonance Imaging are Biomarkers of Neuronal Damage and Inflammation in HIV-negative Cryptococcal Meningoencephalitis.

CNS cryptococcal meningoencephalitis in both HIV positive (HIV+) and HIV negative (HIV-) subjects is associated with high morbidity and mortality despite optimal antifungal therapy. We thus conducted a detailed analysis of the MR imaging findings in 45 HIV- and 11 HIV+ patients to identify imaging findings associated with refractory disease. Ventricular abnormalities, namely ependymitis and choroid plexitis were seen in HIV- but not in HIV+ subjects. We then correlated the imaging findings in a subset of HIV- subjects (n = 17) to CSF levels of neurofilament light chain (NFL), reflective of axonal damage and sCD27, known to best predict the presence of intrathecal T-cell mediated inflammation. We found that ependymitis on brain MRI was the best predictor of higher log(sCD27) levels and choroid plexitis was the best predictor of higher log(NFL) levels. The availability of predictive imaging biomarkers of inflammation and neurological damage in HIV- subjects with CNS cryptococcosis may help gauge disease severity and guide the therapeutic approach in those patients.

Susceptibility to Cryptococcal Meningoencephalitis Associated With Idiopathic CD4+ Lymphopenia and Secondary Germline or Acquired Defects.

Idiopathic CD4+ lymphopenia (ICL) predisposes to opportunistic infections (OIs) but can often remain asymptomatic and does not have a strong association with monogenic mutations. Likewise, cryptococcal meningoencephalitis, the most common OI in ICL, is not strongly associated with monogenic mutations. In this study, we describe 2 patients with ICL plus an additional immune defect: one from an E57K genetic mutation in the nuclear factor-κß essential modulator, and the other with acquired autoantibodies to granulocyte-macrophage colony-stimulating factor. Thus, these cases may exemplify a "multi-hit model" in patients with ICL who acquire OIs.